JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2026
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IF 6.7
Chenchen Nan,Xiaolong Guo,Jiale Cheng,Xin Li,Jun Feng,Jufang Wang,Hongxin Fu
Sabinene, a high-value monoterpene with broad industrial applications, is facing growing demand. To enable sustainable and cost-efficient production, we engineered the yeast Pichia pastoris for sabinene production from methanol. Through systematic metabolic engineering, we implemented a multipronged strategy to optimize sabinene biosynthesis: (1) enhancing precursor supply and synthase activity via fusion proteins (ERG20WW-tNPPS1 and ERG20WW-t37SabS1 (H561F)), (2) redirecting carbon flux through the phosphoketolase-phosphotransacetylase pathway, and (3) upregulating the mevalonate pathway via overexpression of a truncated HMGR. The engineered strain achieved record-high sabinene titers of 3.24 g/L in shake flasks and 6.38 g/L in a 3-L bioreactor. Notably, this yield surpasses those achieved with conventional sugar-based feedstocks in other microbial hosts, underscoring the potential of methanol as a superior carbon source for terpenoid biosynthesis. This work not only establishes P. pastoris as a promising platform for sabinene production, but also paves the way for methanol-based biomanufacturing of diverse natural products.
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Bioresources and Bioprocessing
·
2024
·Vol. 11
·期 1
·1-10
·
IF 4.3
Mei Jianfeng,Han Yanchao,Zhuang Shihang,Yang Zhikai,Yi Yu,Ying Guoqing
Biliverdin, a bile pigment hydrolyzed from heme by heme oxygenase (HO), serves multiple functions in the human body, including antioxidant, anti-inflammatory, and immune response inhibitory activities. Biliverdin has great potential as a clinical drug; however, no economic and efficient production method is available currently. Therefore, the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research. First, the heme oxygenase-1 gene ( HO1 ) encoding the inducible plastidic isozyme from Arabidopsis thaliana , with the plastid transport peptide sequence removed, was recombined into Pichia pastoris GS115 cells. This resulted in the construction of a recombinant P. pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm. After that, the concentration of the inducer methanol, the induction culture time, the pH of the medium, and the concentration of sorbitol supplied in the medium were optimized, resulting in a significant improvement in the yield of HO1. Subsequently, the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride (hemin) into biliverdin. The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30 °C. The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin. Graphical Abstract
研究领域:
生物技术
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药学
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分子生物学
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Bioresources and Bioprocessing
·
2022
·Vol. 9
·期 1
·1-15
·
IF 4.983
Ma Lingling,Liang Xiaolin,Yu Shiqin,Zhou Jingwen
Collagen is a biofunctional protein that has been widely used in many fields, including but not limited to biomedical, cosmetics and skin care, food, and novel materials. Recombinant collagen has great potential as an alternative to collagen extracted from animals because it avoids the immune response, and the yield and properties are stable. However, challenges remain in the industrial application of recombinant collagen, including improving the expression yield, reducing the cost of purification for industry and expanding applications. In this study, a cloning and recombination method was used to heterologously express the recombinant human-like collagen (RHLC) in Pichia pastoris GS115 using the pPIC9k expression vector. The RHLC expression titre was 2.33 g/L via a 5-L fermenter, and the purification was completed within 48 h and was 98% pure. The characteristics of RHLC were investigated. Furthermore, potential applications for RHLC were explored, such as basal collagen sponge preparation, forming films with chitosan and production of collagen hydrolysed peptides. RHLC has various potential applications due to its triple helical structure, thermostability, good biocompatibility and film-forming ability.
研究领域:
生物技术
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生物工程
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材料科学
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药学
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高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2018
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IF 1.338
Jian-yong Zheng,Xing Lan,Xiao-jun Li,Li-juan Huang,Yin-jun Zhang,Zhao Wang
Pichia pastoris expression is a mature and efficient eukaryotic expression system . In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P . pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose™ FF ion-exchange chromatography and phenyl Seflnose™ 6 FF hydrophobic interaction chromatography . The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C 4 ), and the optimum temperature and pH were 40 °C and 8.0, respectively. The purified enzyme activity was inhibited by Zn 2+ and Cu 2+ . Moreover, the K m and V max values were 1 mM and 32.89 mmol/min, respectively.
研究领域:
生物技术
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酶学
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微生物学
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2020
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IF 5.162
Sa Luo,Zhen Qin,Qiming Chen,Liqiang Fan,Lihua Jiang,Liming Zhao
Chitooligosaccharides (COS) are hydrolytic products of chitosan that are essential in functional food, medicine, and other fields due to their biological activities. Commercial COS are often prepared by the hydrolysis of chitosan by chitosanase. In this study, a glycoside hydrolase family 46 cluster B chitosanase from Bacillus amyloliquefaciens (BaCsn46B) was efficiently expressed in Pichia pastoris . The recombinant enzyme was secreted into the culture medium that reached a total extracellular protein concentration of 4.5 g/L with an activity of 8907.2 U/mL in a high cell density fermenter (5 L). The molecular mass of deglycosylated BaCsn46B was 29.0 kDa. Purified BaCsn46B exhibited excellent enzymatic properties, which had high specific activity (2380.5 U/mg) under optimal reaction conditions (55 °C and pH 6.5). BaCsn46B hydrolyzed chitosan yielded a series of COS with different degrees of polymerization by endo-type cleavage. The end hydrolytic products of BaCsn46B were chitobiose and chitotriose, while no monosaccharide yield was evident in the hydrolytic reaction. The excellent secreted expression level and hydrolytic performance make the enzyme a desirable biocatalyst for the industrial preparation of COS.
研究领域:
生物化学
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食品科学
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医学
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高影响因子
Frontiers in Bioengineering and Biotechnology
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2021
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IF 5.89
Miao Ting,Basit Abdul,Liu Junquan,Zheng Fengzhen,Rahim Kashif,Lou Huiqiang,Jiang Wei
Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.
研究领域:
生物技术
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酶学
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生物化学工程
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JOURNAL OF BIOTECHNOLOGY
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2022
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IF 3.595
Wenshuai Zhang,Jianli Zhou,Qiuya Gu,Ruobin Sun,Wenhua Yang,Yu Lu,Congcong Wang,Xiaobin Yu
Chitosanase was widely used in the production of bioactive chitooligosacchride (CHOS) due to their safety, controllability, environmental protection, and biodegradability . Studies showed that the bioactivity of CHOS is closely related to its degree of polymerization. Therefore, the production of ideal polymerized CHOS becomes our primary goal. In this study, the glycosyl hydrolase (GH) family 5 chitosanase was successfully expressed heterologously in Pichia pastoris . After 96 h of high-density fermentation, the chitosanase activity reached 90.62 U·mL −1 , the protein content reached 9.76 mg·mL −1 . When 2% chitosan was hydrolyzed by crude enzyme (20 U/mL), the hydrolysis rate reached 91.2% after 8 h, producing a mixture of CHOS with 2–4 desirable degrees of polymerization (DP). Then, the antioxidant activity of CHOS mixture was investigated, and the results showed that the antioxidant effect was concentration-dependent and had great application potential in the field of nutrition.
研究领域:
生物技术
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酶学
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食品科学
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BIORESOURCE TECHNOLOGY
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2021
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IF 9.642
Juanjuan Ding,Shengping You,Jiaxing Zhang,Hongtao Zhang,Hui Wang,Wei Zhang,Wei Qi,Rongxin Su,Zhimin He
Testosterone (TS) is a critical androgenic steroid that regulates human metabolism and maintains secondary sexual characteristics. The biotransformation from 4-androstene-3,17-done (4-AD) to TS is limited by the poor catalytic activity of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). Herein, we explored the structural characteristics and catalytic mechanism of 17β-HSD3 and adopted the rational design strategy to improve its catalytic activity. Molecular docking and molecular dynamics simulations revealed the substrate-binding pocket and the binding mode of 4-AD to 17β-HSD3. We located the pivotal residues and regulated their hydrophobicity and polarity. The obtained G186R/Y195W variant formed additional electrostatic interaction and hydrogen bond with 4-AD, increasing the binding affinity between the variant and 4-AD. Therefore, the G186R/Y195W variant produced 3.98 g/L of TS, which increased to 297%. The combination of structural and mechanism resolution drives the implementation of the rational design strategy, which provides guidance for bioproduction of TS catalyzed by 17β-HSD3.
研究领域:
生物化学
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酶工程
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生物技术
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PROTEIN EXPRESSION AND PURIFICATION
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2015
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IF 1.695
Xin Chen,Yong-hao Hu,Wei-di Chen,Wen-di Li,Zi-chao Huang,Yun Li,Yu-wen Luo,Yu-xia Huang,Yi-tai Chen,Kai Wang,Ling Li
Background Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation. Objective To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities. Methods Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZαA) and constitutive (pGAPZαA) vectors in order to create Pichia pastori s GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE. Results After 120 h of fermentation, the yield of constitutive plectasin (370 μg/ml) was much lower than that from inducible vector (880 μg/ml) ( P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield ( P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 μg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 μg/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P ＞ 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass. Conclusions Although P. pastoris GS115 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.
研究领域:
分子生物学
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微生物学
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生物技术
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高影响因子
Microorganisms
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2023
·Vol. 12
·期 1
·38
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IF 4.5
Kun Geng,Ying Lin,Xueyun Zheng,Cheng Li,Shuting Chen,He Ling,Jun Yang,Xiangyu Zhu,Shuli Liang
Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) inSaccharomyces cerevisiaecatalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study,ADH1was successfully expressed inPichia pastorisGS115 based on codon optimization. Then, the expression level ofADH1was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.Keywords:alcohol dehydrogenase I;Pichia pastoris;cofactor regeneration;multicopy;acetaldehyde;wine
研究领域:
微生物学
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生物技术
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食品科学
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高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2020
·Vol. 44
·期 1
·67-79
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IF 2.419
Zhou Hai-Yan,Zhou Jian-Bao,Yi Xiao-Nan,Wang Yan-Mei,Xue Ya-Ping,Chen De-Shui,Cheng Xin-Ping,Li Mian,Wang Hong-Yan,Chen Kai-Qian,Liu Zhi-Qiang,Zheng Yu-Guo
To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55–75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0–5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0–6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt ( n = approx. 500) (CMC) and β - d -glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters V max , K m , and K cat / K m values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β - d -glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.
研究领域:
生物化学
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生物技术
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酶学
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微生物学
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高影响因子
Microbial Cell Factories
·
2019
·Vol. 18
·期 1
·1-12
·
IF 4.402
Li Yi-Min,Gao Jiao-Qi,Pei Xu-Ze,Du Cong,Fan Chao,Yuan Wen-Jie,Bai Feng-Wu
Background l-Alanyl-l-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln.Results In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles.Conclusion sCharacterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.
研究领域:
生物技术
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酶学
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药物化学
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合成生物学
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PubMed
高影响因子
危险品化学品经营许可证（带存储）