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
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2024
·
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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ACS Sustainable Chemistry & Engineering
·
2024
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IF 7.1
Feng Xiao,Dongfang Li,Yingjia Pan,Binying Lv,Jucan Gao,Yimeng Zuo,Lei Huang,Jiazhang Lian
Cholesterol sulfate (CS), a sulfated derivative of cholesterol, has important applications in the medical field, because of its extensive biological and pharmacological activities. As the supply of CS heavily relies on chemical conversion of cholesterol extracted from wool grease, there is growing interest in engineering microbial cell factories for large-scale production of CS. This study reports microbial production of CS in the yeast, Komagataella phaffii (also known as Pichia pastoris), for the first time. Through combinatorial metabolic engineering strategies, including blocking the competing branches, overexpressing the rate-limiting mevalonate pathway genes, manipulating lipid metabolism, and enhancing the supply of key cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), CS production was increased more than 6.8-fold when compared with the parent strain. The optimal strain (CHS0505) produced CS with titers reaching 249 mg/L in a shake flask and 545 mg/L in a fed-batch fermenter, highlighting the potential of K. phaffii as a cell factory for steroid production. More importantly, the development of a CS-producing cell factory will advance microbial production of glycosaminoglycans and other sulfated natural products via synthetic biology.
研究领域:
生物工程
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合成生物学
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医药工程
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Nature Synthesis
·
2026
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IF 22.6
Wang Shuai,Yang Liu,Li Zhixuan,Ye Hui,Teng Fei,Yang Jun,Ye Chunting,Fan Dexun,Han Shuangyan
Methanol is a promising C1 feedstock for bioproduction but its cytotoxicity limits applications. Here we enhanced Komagataella phaffii GS115 methanol tolerance to 10–13% (v/v) by overexpressing four genes (phosphatidylethanolamine methyltransferase ( PET2 ), phosphatidylserine synthase ( PSA ), PAS_chr1-4_017 8 ( 0178 ) and PAS_chr2-2_0267 ( 0267 )) (S33 strain). Our results show that, under high methanol stress, S33 undergoes membrane lipid remodelling-driven metabolic reprogramming, with central carbon flux being redistributed from the pentose phosphate pathway and citric acid cycle towards glycolysis. Within this framework, 0178 encodes a vacuolar transporter of sulfur-containing amino acids, which we propose supports phospholipid methylation and redox balance. Meanwhile, 0267 encodes a peroxisomal thiolase-degrading long‑chain fatty acyl‑CoAs, limiting toxic long-chain sphingolipid accumulation, maintaining membrane stability. These genes enable S33 to achieve lipid titre of approximately 40 g l −1 under non-sterile fed‑batch fermentation conditions. This work positions S33 as a methanol-tolerant K. phaffii chassis for further strain and process development.
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Biotechnology Journal
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2025
·Vol. 20
·期 6
·e70036
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IF 3.1
Shupeng Ruan,Aoxue Wang,Hongyi Zou,Ying Lin,Lei Ye,Shuli Liang
The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine-tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7-tRNA-sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet-on induction system, split-T7 system, and RNA cleavage processing by HH-HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self-responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.
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Nature Communications
·
2026
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IF 18.1
Zhu Xiangyu,Lin Jiawen,Liang Shuli,Qiu Chenyi,Jiang Yuchen,Liu Hengrui,Zhu Kun,Wang Qi,Du Chenfeng,Li Yonghao,Mao Yuanhui,Zheng Huanbin,Liu Chengan,Zhao Yeyao,An Meirong,Wu Yifeng,Liu Jun,Zhang Xinying,Lin Ying
Pyomelanin has been extensively applied in various fields. However, the yield of pyomelanin isolated from natural producers is low. Engineering microbial biosynthesis is considered a sustainable and economically feasible alternative. We utilize engineered Komagataella phaffii to increase the yield of homogentisic acid by 66-fold by balancing three different biosynthetic modules and further synthesizing pyomelanin by oxidative polymerization. During this process, we establish a pyomelanin color screening system and apply this system to perform directed evolution of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase and semirational design modification of hydroxyphenylpyruvate dioxygenase. We report the difference of pyomelanin between oxidative polymerization under alkaline conditions and that under laccase treatment. After high-density fermentation in a 5-L fermenter for 204 h, strain Pyo29 achieve the highest titer (70.5 ± 0.7 g/L). Finally, we demonstrate the potential application value of pyomelanin. This study provides a feasible solution for high-yield pyomelanin biosynthesis with substantial industrial production.
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Biotechnology Journal
·
2026
·Vol. 21
·期 8
·e70288
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IF 3.7
Yanxuan Wu,Chengan Liu,Shuli Liang,Yuchen Jiang,Xinying Zhang,Yuqi Zhou,Ying Lin
Retinol, a derivative of vitamin A with potent antioxidant and therapeutic properties, is in high market demand. In response to the low productivity of conventional methods, metabolic engineering has been explored for microbial retinol production. However, systematic engineering strategies for high-level retinol synthesis in Komagataella phaffii remain limited. In this study, the methylotrophic yeast K. phaffii was developed as an engineered chassis for efficient de novo retinol biosynthesis. Based on a previously constructed β-carotene-producing strain, β-carotene-15,15′-dioxygenase (Blh) and retinol dehydrogenase (RDH12) were screened and introduced to establish the synthetic pathway of retinol. To increase precursor supply, key genes in the β-carotene biosynthetic pathway were overexpressed. The mevalonate (MVA) pathway was further optimized, and central carbon metabolism was reprogrammed to enhance metabolic flux toward retinol. Transport engineering was also performed to improve retinol secretion. Several candidate transporters were overexpressed, and the protein encoded by chr1-4_0619 in K. phaffii was identified as an endogenous retinol transporter. The final engineered strain produced 3.38 g/L retinol with BHT supplementation in fed-batch fermentation using a 1.5 L bioreactor. This work represents de novo microbial synthesis of retinol from a one-carbon feedstock, demonstrating the formidable potential of K. phaffii as a sustainable chassis for retinol production.
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CHEMICAL ENGINEERING JOURNAL
·
2025
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IF 13.2
Guang Li,Xiangpeng Yang,Lei Huang,Jiaying Wang,Xiangrui Wu,Benhong Xu,Liang Li,Hongbin Yuan,Yuhuan Liu,Jinwen Wang,Lichuang Cao
Despite its longstanding use in recombinant protein production, the fundamental principles governing signal peptide (SP) functionality in Komagataella phaffii ( K. phaffii ) remain poorly understood. In this study, 64 endogenous SPs were identified from K. phaffii genome and were combined with 52 high-performance exogenous SPs from literature. This SP library was employed to optimize secretion of β-galactosidase Bgal1–3-Y497F, an efficient biocatalyst for producing galactooligosaccharide-enriched and low-lactose dairy products. The characterization identified 35 new SPs, expanding the repertoire of SPs that outperformed the conventional α-mating factor (α-MF) SP by 30 %. The top performers, SP25 and W1, mediated 4.0- and 5.5-fold higher extracellular enzyme activity than α-MF SP, with similar enhancements (2.0–5.5-fold) observed for heterologous proteins β-galactosidase BgaD-D and plastic-degrading enzyme LCC-ICCG. A few key sequence features for efficient secretion were revealed. For N-regions, endogenous SPs preferred +1 charge, whereas exogenous SPs preferred +3 charges. For H-region, both SP types showed bell-curve dependencies (extracellular enzyme activity plotted against hydrophobicity) with a consistent optimal window (hydrophobicity scores = 40–55) that provides a general guideline for SP selection and engineering in this host. Implementing an integrated engineering strategy (W1-based SP engineering, multi-copy strain selection, chaperone co-expression, and cultivation condition optimization), a 17.9-fold production increase was achieved. Scale-up to 5-L bioreactor cultivation yielded 7.0 g/L, the highest reported titer of prokaryotic β-galactosidase in K. phaffii . This work provides both fundamental insights into SP features for efficient protein secretion in K. phaffii and a high-performance industrial strain for dairy processing applications.
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CHEMICAL ENGINEERING JOURNAL
·
2025
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IF 13.2
Haibo Xiong,Xiaogang Yang,Weijiao Zhang,Litao Hu,Gege Liu,Guocheng Du,Jian Chen,Jianghua Li,Ruirui Xu,Zhen Kang
Chondroitin sulfate (CS), which is primarily extracted from animal tissues, has been widely used for maintaining joint and cartilage health. Despite a few innovations in CS biosynthesis, low titer or sulfation degree values remain bottlenecks for its potential applications. We developed robust Komagataella phaffii cell factories for producing highly sulfated chondroitin sulfate A (CSA). First, 82.3 mg/L CSA with a sulfation degree of 5.5 % was produced through the reconstruction of the chondroitin synthesis pathway, active expression of chondroitin 4- O -sulfotransferase (C4ST), and addition of adequate sulfates. Further overexpression of the sulfate transporter SUL1 elevated the sulfation level to 18.6 %. After revealing the quantitative relationship between chondroitin synthesis and its sulfation, the expression of C4ST improved with multicopy integration, and the sulfation degree of CSA increased to 57.7 %. Thereafter, a 3′-phosphoadenosine-5′-phosphosulfate regeneration system was introduced and optimized, which further elevated the sulfation degree to 90.3 %. Finally, a growth-coupled feeding approach was adopted to control the concentration of heavy metal ions, and the engineered strain CS10 produced 1.15 g/L (10.92 mg/g DCW) CSA with a sulfation degree of 96 % in fed-batch cultures. Our study provides an attractive alternative for the biomanufacturing of highly sulfated CSA. Furthermore, the engineered K. phaffii cell factories could be used for producing sulfated compounds of interest, such as heparin, and anticoagulant peptides.
研究领域:
生物技术
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合成生物学
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微生物学
·
生物工程
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高影响因子
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