Pub Date : 2025-01-26DOI: 10.1016/j.synbio.2025.01.007
Tianyu Dong , Yujie Shu , Ying Wang , Mingdong Yao , Wenhai Xiao
Yarrowia lipolytica, a safe yeast, efficiently metabolizes lipids for the production of food additives and agricultural products. Boosting its growth and lipid utilization capabilities is crucial to enhancing the overall efficiency Y. lipolytica. Herein, an integrated strategy was implemented to enhance lipid uptake, accumulation and metabolism and systematically promote the growth and lipid utilization of the commonly used Y. lipolytica Po1f strain. The engineered strain had a specific growth rate of 0.32 h−1 and a lipid content of 67.66 % (g/g DCW), which were 54 % and 26 % greater than those of the original strain. β-Carotene was used to verify the production of lipophilic natural compounds, and the highest yield was obtained 48 h earlier using the engineered strain compared to the original strain when consuming same carbon source. These findings show promise in using engineered Y. lipolytica for rapid growth and improved lipid utilization to boost efficiency of lipophilic product production.
{"title":"An engineered Yarrowia lipolytica with rapid growth and efficient lipid utilization","authors":"Tianyu Dong , Yujie Shu , Ying Wang , Mingdong Yao , Wenhai Xiao","doi":"10.1016/j.synbio.2025.01.007","DOIUrl":"10.1016/j.synbio.2025.01.007","url":null,"abstract":"<div><div><em>Yarrowia lipolytica</em>, a safe yeast, efficiently metabolizes lipids for the production of food additives and agricultural products. Boosting its growth and lipid utilization capabilities is crucial to enhancing the overall efficiency <em>Y. lipolytica</em>. Herein, an integrated strategy was implemented to enhance lipid uptake, accumulation and metabolism and systematically promote the growth and lipid utilization of the commonly used <em>Y. lipolytica</em> Po1f strain. The engineered strain had a specific growth rate of 0.32 h<sup>−1</sup> and a lipid content of 67.66 % (g/g DCW), which were 54 % and 26 % greater than those of the original strain. <em>β</em>-Carotene was used to verify the production of lipophilic natural compounds, and the highest yield was obtained 48 h earlier using the engineered strain compared to the original strain when consuming same carbon source. These findings show promise in using engineered <em>Y. lipolytica</em> for rapid growth and improved lipid utilization to boost efficiency of lipophilic product production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 495-503"},"PeriodicalIF":4.4,"publicationDate":"2025-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143349164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-25DOI: 10.1016/j.synbio.2025.01.008
Lanmei Zhao , Mengxue Sun , Can Lyu , Long Meng , Jian Liu , Bo Wang
<div><div>This study presented new insights into the sustainable conversion of total petroleum hydrocarbon (TPHC) into polyhydroxyalkanoates (PHAs) using wetland microbial fuel cells (WMFCs). The main innovations included the following two points: (1) The integration of bioelectricity generation with efficient PHA production further underscored the potential of electroactive biofilms as a sustainable platform for simultaneous TPHC biotransformation, bioelectricity recovery and PHA production. (2) The interactive dynamics of PHAs, metabolites, extracellular polymeric substances (EPS) and microorganisms during the formation and stabilization of electroactive biofilms provided novel insights into microbial strategies for carbon utilization. As the electroactive biofilm formed and stabilized, the current density enhanced significantly from 0 to 101 mA m<sup>−</sup><sup>2</sup>, then stabilized, and finally dropped to 3.51 mA m<sup>−2</sup>. Similarly, the power density showed a trend of increasing in the initial stage, maintaining in the middle stage, and then descending in the later stage. The production of six types of PHAs was identified: poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxyvalerate) [P(3HV)], poly(3-hydroxybutyrate-<em>co</em>-3-hydroxyvalerate) [P(3HB-<em>co</em>-3HV)], poly[(R)-3-hydroxybutyrate-<em>co</em>-(R)-3-hydroxyhexanoate] [P(3HB-<em>co</em>-3HHX)], poly(3-hydroxyhexadecanoate) [P(3HHD)] and poly(3-hydroxyoctadecanoate) [P(3HOD)], highlighting the metabolic flexibility of electroactive biofilms. The total PHA content was initially undetectable (days 0–4), gradually increased (days 4–28), rose rapidly (days 28–48), gradually increased and descended (days 48–68). The maximum PHA content of 0.664 g g⁻<sup>1</sup> DCW achieved highlighted the dual functionality of WMFCs in bioelectricity production and PHA biosynthesis, distinguishing it from conventional MFC applications. The TPHC biodegradation ratio demonstrated a gradual increase (days 0–28), with a more pronounced rise (days 28–48), and a gradual rise to 76.1 % (days 48–68). Throughout the process, the metabolite volatile fatty acids (VFAs) produced were primarily acetate, propionate, butyrate and valerate. The trend of VFA production from days 0–56 closely followed that of TPHC biodegradation. The trend of tyrosine/tryptophan proteins in EPS was aligned with that of biofilm thickness. The strong correlation between the increase in the biofilm thickness and the intensity and peak height of tyrosine/tryptophan proteins during the first 20 days suggested that these proteins were integral to the structural integrity of the biofilms, and from days 20–64, the minimal variation in their intensity and peak height indicated that the biofilms had reached a relatively stable state. The biofilms in turn provided a stable microbial substrate and energetic support for the subsequent efficient synthesis of PHA. During the early phase, the dual-function bacteria, such as <em>Pseudomonas</
{"title":"Polyhydroxyalkanoate production during electroactive biofilm formation and stabilization in wetland microbial fuel cells for petroleum hydrocarbon bioconversion","authors":"Lanmei Zhao , Mengxue Sun , Can Lyu , Long Meng , Jian Liu , Bo Wang","doi":"10.1016/j.synbio.2025.01.008","DOIUrl":"10.1016/j.synbio.2025.01.008","url":null,"abstract":"<div><div>This study presented new insights into the sustainable conversion of total petroleum hydrocarbon (TPHC) into polyhydroxyalkanoates (PHAs) using wetland microbial fuel cells (WMFCs). The main innovations included the following two points: (1) The integration of bioelectricity generation with efficient PHA production further underscored the potential of electroactive biofilms as a sustainable platform for simultaneous TPHC biotransformation, bioelectricity recovery and PHA production. (2) The interactive dynamics of PHAs, metabolites, extracellular polymeric substances (EPS) and microorganisms during the formation and stabilization of electroactive biofilms provided novel insights into microbial strategies for carbon utilization. As the electroactive biofilm formed and stabilized, the current density enhanced significantly from 0 to 101 mA m<sup>−</sup><sup>2</sup>, then stabilized, and finally dropped to 3.51 mA m<sup>−2</sup>. Similarly, the power density showed a trend of increasing in the initial stage, maintaining in the middle stage, and then descending in the later stage. The production of six types of PHAs was identified: poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxyvalerate) [P(3HV)], poly(3-hydroxybutyrate-<em>co</em>-3-hydroxyvalerate) [P(3HB-<em>co</em>-3HV)], poly[(R)-3-hydroxybutyrate-<em>co</em>-(R)-3-hydroxyhexanoate] [P(3HB-<em>co</em>-3HHX)], poly(3-hydroxyhexadecanoate) [P(3HHD)] and poly(3-hydroxyoctadecanoate) [P(3HOD)], highlighting the metabolic flexibility of electroactive biofilms. The total PHA content was initially undetectable (days 0–4), gradually increased (days 4–28), rose rapidly (days 28–48), gradually increased and descended (days 48–68). The maximum PHA content of 0.664 g g⁻<sup>1</sup> DCW achieved highlighted the dual functionality of WMFCs in bioelectricity production and PHA biosynthesis, distinguishing it from conventional MFC applications. The TPHC biodegradation ratio demonstrated a gradual increase (days 0–28), with a more pronounced rise (days 28–48), and a gradual rise to 76.1 % (days 48–68). Throughout the process, the metabolite volatile fatty acids (VFAs) produced were primarily acetate, propionate, butyrate and valerate. The trend of VFA production from days 0–56 closely followed that of TPHC biodegradation. The trend of tyrosine/tryptophan proteins in EPS was aligned with that of biofilm thickness. The strong correlation between the increase in the biofilm thickness and the intensity and peak height of tyrosine/tryptophan proteins during the first 20 days suggested that these proteins were integral to the structural integrity of the biofilms, and from days 20–64, the minimal variation in their intensity and peak height indicated that the biofilms had reached a relatively stable state. The biofilms in turn provided a stable microbial substrate and energetic support for the subsequent efficient synthesis of PHA. During the early phase, the dual-function bacteria, such as <em>Pseudomonas</","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 474-483"},"PeriodicalIF":4.4,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143161007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-23DOI: 10.1016/j.synbio.2025.01.005
Kaidi Chen , Gulikezi Maimaitirexiati , Qiannan Zhang , Yi Li , Xiangjian Liu , Hongting Tang , Xiang Gao , Bo Wang , Tao Yu , Shuyuan Guo
The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of P. pastoris, especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three sgRNA processing cassettes, including a tRNA-sgRNA-tRNA (tgt) array, HgH structure and tRNA-sgRNA-HDV (tgH) structure. Furthermore, the dHgH structure (double HgH) enabled one-step dual-gene disruption and multi-gene integration. The efficiency of dual-site knockout ranged from 60 % to 100 %, with functional genes knockout achieving approximately 60 % (Δaox1Δgut1), while dual neutral sites knockout reached 100 %. Finally, we applied the system for one-step production of fatty acids and 5-hydroxytryptophan. The yield of FFAs reached 23 mg/L/μg protein/OD, while the yield of 5-hydroxytryptophan was 13.3 mg/L. The system will contribute to the application of P. pastoris as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization.
{"title":"CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris","authors":"Kaidi Chen , Gulikezi Maimaitirexiati , Qiannan Zhang , Yi Li , Xiangjian Liu , Hongting Tang , Xiang Gao , Bo Wang , Tao Yu , Shuyuan Guo","doi":"10.1016/j.synbio.2025.01.005","DOIUrl":"10.1016/j.synbio.2025.01.005","url":null,"abstract":"<div><div>The important methylotrophic yeast <em>Pichia pastoris</em> has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of <em>P. pastoris</em>, especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three sgRNA processing cassettes, including a tRNA-sgRNA-tRNA (tgt) array, HgH structure and tRNA-sgRNA-HDV (tgH) structure. Furthermore, the dHgH structure (double HgH) enabled one-step dual-gene disruption and multi-gene integration. The efficiency of dual-site knockout ranged from 60 % to 100 %, with functional genes knockout achieving approximately 60 % (<em>Δaox1Δgut1</em>), while dual neutral sites knockout reached 100 %. Finally, we applied the system for one-step production of fatty acids and 5-hydroxytryptophan. The yield of FFAs reached 23 mg/L/μg protein/OD, while the yield of 5-hydroxytryptophan was 13.3 mg/L. The system will contribute to the application of <em>P. pastoris</em> as an attractive cell factory for multiplexed compound biosynthesis and will serve as a valuable tool for enhancing one-carbon (C1) bio-utilization.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 484-494"},"PeriodicalIF":4.4,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143312236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-22DOI: 10.1016/j.synbio.2025.01.003
Yue Huang , Shao Jia , Ying Lin , Jialiang Wang , Luyuan Nong , Lei Ye , Shuli Liang
Patatin, a prominent food protein derived from potatoes, is renowned for its exceptional nutritional value. Patatin has been characterized for its diverse physiological attributes, including esterase activity, antioxidative properties, cholesterol-lowering effects, and high lysine content, alongside notable physicochemical traits such as foaming, emulsification, and gelation capabilities. Conventional methods for patatin extraction are fraught with inefficiencies, elevated costs, and detrimental impacts on protein structural and functional integrity. Herein, we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre (g/L) in a 5-Litre bioreactor, which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far. In this study, we innovatively refined the endogenous promoter PCAT1, and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter. Furthermore, crucial nodes for patatin heterologous expression in yeast were identified, substantially curtailing the production costs associated with patatin synthesis.
{"title":"High-efficiency patatin expression strategies in Komagataella phaffii (Pichia pastoris): Expression cassette toolbox and regulation of protein secretion","authors":"Yue Huang , Shao Jia , Ying Lin , Jialiang Wang , Luyuan Nong , Lei Ye , Shuli Liang","doi":"10.1016/j.synbio.2025.01.003","DOIUrl":"10.1016/j.synbio.2025.01.003","url":null,"abstract":"<div><div>Patatin, a prominent food protein derived from potatoes, is renowned for its exceptional nutritional value. Patatin has been characterized for its diverse physiological attributes, including esterase activity, antioxidative properties, cholesterol-lowering effects, and high lysine content, alongside notable physicochemical traits such as foaming, emulsification, and gelation capabilities. Conventional methods for patatin extraction are fraught with inefficiencies, elevated costs, and detrimental impacts on protein structural and functional integrity. Herein, we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness <em>Komagataella phaffii</em> as the expression host and achieved an expression level of 3.2 g per litre (g/L) in a 5-Litre bioreactor, which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far. In this study, we innovatively refined the endogenous promoter P<sub><em>CAT1</em></sub>, and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional <em>AOX1</em> promoter. Furthermore, crucial nodes for patatin heterologous expression in yeast were identified, substantially curtailing the production costs associated with patatin synthesis.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 463-473"},"PeriodicalIF":4.4,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143161006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-22DOI: 10.1016/j.synbio.2025.01.004
Yi-Tong Yao , Xiao Zhang , Chen-Yu Wang, Yu-He Zhang, Da-Wei Li, Wei-Dong Yang, Hong-Ye Li, Li-Gong Zou
Longifolene (C15H24) is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications. Traditional extraction methods from plants suffer from low titer and lengthy production cycles, while chemical synthesis is hampered by the compound's complex structure, leading to high costs and insufficient market supply. This study aimed to develop a microbial cell factory for enhanced longifolene production. The strategy involved integrating longifolene synthase from Pinus sylvestris (PsTPS) into Yarrowia lipolytica and employing multiple metabolic engineering approaches. Initially, key genes in the mevalonate (MVA) pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis. Subsequently, protein engineering techniques were applied to optimize PsTPS (tPsTPS) for improved catalytic efficiency. Furthermore, co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of PsTPS. The introduction of the isopentenol utilization pathway (IUP) further augmented the supply of C5 substrate. By optimizing the culture conditions, including a reduction in culture temperature, the efflux of longifolene was increased, and the dissolved oxygen levels were enhanced to promote the growth of the strain. These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks. This study not only demonstrates the feasibility of enhancing sesquiterpene production in Y. lipolytica but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.
{"title":"Optimizing longifolene production in Yarrowia lipolytica via metabolic and protein engineering","authors":"Yi-Tong Yao , Xiao Zhang , Chen-Yu Wang, Yu-He Zhang, Da-Wei Li, Wei-Dong Yang, Hong-Ye Li, Li-Gong Zou","doi":"10.1016/j.synbio.2025.01.004","DOIUrl":"10.1016/j.synbio.2025.01.004","url":null,"abstract":"<div><div>Longifolene (C<sub>15</sub>H<sub>24</sub>) is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications. Traditional extraction methods from plants suffer from low titer and lengthy production cycles, while chemical synthesis is hampered by the compound's complex structure, leading to high costs and insufficient market supply. This study aimed to develop a microbial cell factory for enhanced longifolene production. The strategy involved integrating longifolene synthase from <em>Pinus sylvestris</em> (<em>PsTPS</em>) into <em>Yarrowia lipolytica</em> and employing multiple metabolic engineering approaches. Initially, key genes in the mevalonate (MVA) pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis. Subsequently, protein engineering techniques were applied to optimize <em>PsTPS</em> (t<em>PsTPS</em>) for improved catalytic efficiency. Furthermore, co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of <em>PsTPS</em>. The introduction of the isopentenol utilization pathway (IUP) further augmented the supply of C5 substrate. By optimizing the culture conditions, including a reduction in culture temperature, the efflux of longifolene was increased, and the dissolved oxygen levels were enhanced to promote the growth of the strain. These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks. This study not only demonstrates the feasibility of enhancing sesquiterpene production in <em>Y. lipolytica</em> but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 433-441"},"PeriodicalIF":4.4,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143161057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-17DOI: 10.1016/j.synbio.2025.01.002
Chun Su , Nguyen-Quang Tuan , Wen-Hua Li , Jin-Hua Cheng , Ying-Yu Jin , Soon-Kwang Hong , Hyun Lee , Mallique Qader , Larry Klein , Gauri Shetye , Guido F. Pauli , Scott G. Flanzblau , Sang-Hyun Cho , Xin-Qing Zhao , Joo-Won Suh
Streptomyces sp. MJM3502 is a promising producer of rufomycins, which are a class of potent anti-tuberculosis lead compounds. Although the structure, activity, and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied, a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of Streptomyces sp. MJM3502, and its metabolic engineering has not yet been reported. This study established the genetic manipulation platform for the strain. Using CRISPR/Cas9-based technology to in-frame insert the strong kasO∗p promoter upstream of the rufB and rufS genes of the rufomycin BGC, we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold, respectively. Furthermore, designing recombinant strains by inserting the kasO∗p promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives. These findings provide the basis for enhancing the production of valuable natural compounds in Streptomyces and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.
{"title":"Enhancing rufomycin production by CRISPR/Cas9-based genome editing and promoter engineering in Streptomyces sp. MJM3502","authors":"Chun Su , Nguyen-Quang Tuan , Wen-Hua Li , Jin-Hua Cheng , Ying-Yu Jin , Soon-Kwang Hong , Hyun Lee , Mallique Qader , Larry Klein , Gauri Shetye , Guido F. Pauli , Scott G. Flanzblau , Sang-Hyun Cho , Xin-Qing Zhao , Joo-Won Suh","doi":"10.1016/j.synbio.2025.01.002","DOIUrl":"10.1016/j.synbio.2025.01.002","url":null,"abstract":"<div><div><em>Streptomyces</em> sp. MJM3502 is a promising producer of rufomycins, which are a class of potent anti-tuberculosis lead compounds. Although the structure, activity, and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied, a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of <em>Streptomyces</em> sp. MJM3502, and its metabolic engineering has not yet been reported. This study established the genetic manipulation platform for the strain. Using CRISPR/Cas9-based technology to in-frame insert the strong <em>kasO∗p</em> promoter upstream of the <em>rufB</em> and <em>rufS</em> genes of the rufomycin BGC, we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold, respectively. Furthermore, designing recombinant strains by inserting the <em>kasO∗p</em> promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives. These findings provide the basis for enhancing the production of valuable natural compounds in <em>Streptomyces</em> and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 421-432"},"PeriodicalIF":4.4,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143160997","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-07DOI: 10.1016/j.synbio.2025.01.001
Wenqi Ding , Tong Lin , Yun Yang , Wen-Wei Li , Shaoan Cheng , Hao Song
The primary limitation to the practicability of electroactive microorganisms in bioelectrochemical systems lies in their low extracellular electron transfer (EET) efficiency. The proton motive force (PMF) represents the electrochemical gradient of protons generated by electron transport and proton pumping across the cytoplasmic membrane, serving as a crucial energy transfer pathway in bacterial membranes. Nevertheless, the impact of PMF on the EET efficiency remains ambiguous, while the microbial rhodopsin offers a simple and efficient avenue for non-photosynthetic cells to harness PMF. Here, we studied the function of three microbial rhodopsins (Arch, Mac, and cR-1) in facilitating EET via their heterologous expression in S. oneidensis, a model electroactive microorganism. Among these, the recombinant strain expressing rhodopsin cR-1 exhibited the highest output power density of 0.87 W/m2, 3.49-fold increase over the wild-type S. oneidensis MR-1. Our further transcriptomics analyses of the energy and materials metabolism of strain cR-1 showed that the underlying mechanism of enhanced EET efficiency was resulted from heterologous expression of the light-driven proton pump. The results suggested that strain cR-1 effectively expels protons to generate additional PMF and provide extra ATP supply to the cells, which facilitated lactate uptake and utilization, thus enhancing electrons generation in cells. This augmented intracellular electron pool capacity ultimately resulted in enhancement of EET rate and power generation efficiency of the recombinant S. oneidensis.
{"title":"Proton motive force generated by microbial rhodopsin promotes extracellular electron transfer","authors":"Wenqi Ding , Tong Lin , Yun Yang , Wen-Wei Li , Shaoan Cheng , Hao Song","doi":"10.1016/j.synbio.2025.01.001","DOIUrl":"10.1016/j.synbio.2025.01.001","url":null,"abstract":"<div><div>The primary limitation to the practicability of electroactive microorganisms in bioelectrochemical systems lies in their low extracellular electron transfer (EET) efficiency. The proton motive force (PMF) represents the electrochemical gradient of protons generated by electron transport and proton pumping across the cytoplasmic membrane, serving as a crucial energy transfer pathway in bacterial membranes. Nevertheless, the impact of PMF on the EET efficiency remains ambiguous, while the microbial rhodopsin offers a simple and efficient avenue for non-photosynthetic cells to harness PMF. Here, we studied the function of three microbial rhodopsins (Arch, Mac, and cR-1) in facilitating EET via their heterologous expression in <em>S. oneidensis</em>, a model electroactive microorganism. Among these, the recombinant strain expressing rhodopsin cR-1 exhibited the highest output power density of 0.87 W/m<sup>2</sup>, 3.49-fold increase over the wild-type <em>S. oneidensis</em> MR-1. Our further transcriptomics analyses of the energy and materials metabolism of strain cR-1 showed that the underlying mechanism of enhanced EET efficiency was resulted from heterologous expression of the light-driven proton pump. The results suggested that strain cR-1 effectively expels protons to generate additional PMF and provide extra ATP supply to the cells, which facilitated lactate uptake and utilization, thus enhancing electrons generation in cells. This augmented intracellular electron pool capacity ultimately resulted in enhancement of EET rate and power generation efficiency of the recombinant <em>S. oneidensis</em>.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 410-420"},"PeriodicalIF":4.4,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11786069/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143081010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.synbio.2024.10.008
Li Liang, Xu Shan-Shan, Jiang Yan-Jun
Ergothioneine (ERG), a rare natural thio-histidine derivative with potent antioxidant properties and diverse biological functions, is widely utilized in food processing, cosmetics, pharmaceuticals, and nutritional supplements. Current bioproduction methods for ERG primarily depend on fermenting edible mushrooms. However, with the advancement in synthetic biology, an increasing number of genetically engineered microbial hosts have been developed for ERG production, including Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Given the involvement of multiple precursor substances in ERG synthesis, it is crucial to employ diverse strategies to regulate the metabolic flux of ERG synthesis. This review comprehensively evaluates the physiological effects and safety considerations associated with ERG, along with the recent advancements in catalytic metabolic pathway for ERG production using synthetic biology tools. Finally, the review discusses the challenges in achieving efficient ERG production and the strategies to address these challenges using synthetic biology tools. This review provides a literature analysis and strategies guidance for the further application of novel synthetic biology tools and strategies to improve ERG yield.
{"title":"Ergothioneine biosynthesis: The present state and future prospect","authors":"Li Liang, Xu Shan-Shan, Jiang Yan-Jun","doi":"10.1016/j.synbio.2024.10.008","DOIUrl":"10.1016/j.synbio.2024.10.008","url":null,"abstract":"<div><div>Ergothioneine (ERG), a rare natural thio-histidine derivative with potent antioxidant properties and diverse biological functions, is widely utilized in food processing, cosmetics, pharmaceuticals, and nutritional supplements. Current bioproduction methods for ERG primarily depend on fermenting edible mushrooms. However, with the advancement in synthetic biology, an increasing number of genetically engineered microbial hosts have been developed for ERG production, including <em>Escherichia coli</em>, <em>Saccharomyces cerevisiae</em>, and <em>Corynebacterium glutamicum</em>. Given the involvement of multiple precursor substances in ERG synthesis, it is crucial to employ diverse strategies to regulate the metabolic flux of ERG synthesis. This review comprehensively evaluates the physiological effects and safety considerations associated with ERG, along with the recent advancements in catalytic metabolic pathway for ERG production using synthetic biology tools. Finally, the review discusses the challenges in achieving efficient ERG production and the strategies to address these challenges using synthetic biology tools. This review provides a literature analysis and strategies guidance for the further application of novel synthetic biology tools and strategies to improve ERG yield.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 314-325"},"PeriodicalIF":4.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11664081/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.synbio.2024.11.006
Jia-Wei Ren , Jin-Peng Zhang , Zi-Lun Mei , Jia-Yi Shao , Guo-Qiang Xu , Hui Li , Jin-Song Gong , Xiao-Mei Zhang , Jin-Song Shi , Xiao-juan Zhang , Zheng-hong Xu
The primary function of terminators is to terminate transcription in gene expression. Although some studies have suggested that terminators also contribute positively to upstream gene expression, the extent and underlying mechanism of this effect remain largely unexplored. Here, the correlation between terminating strength and upstream mRNA stability was investigated by constructing a terminator mutation library through randomizing 5 nucleotides, assisted by FlowSeq technology, terminator variants were categorized based on the downstream fluorescence intensity, followed by high-throughput sequencing. To examine the impact of terminators on mRNA stability, the abundance of downstream gene transcripts for each terminator variant was quantified through cDNA sequencing. The results revealed that the transcript abundance controlled by strong terminators was, on average 2.2 times greater than those controlled by weak terminators on average. Moreover, several distinct features could be ascribed to high relative abundance of upstream gene transcript, including a high GC content at the base region of hairpin, and a high AT content in downstream of the U-tract. Additionally, these terminators showed a free energy between −28 and −22 kcal/mol, and a stem length of 14 nt. Finally, these features ascribed the upstream beneficial terminator were validated across various expression systems. By incorporating the optimal terminator downstream of RSF, GSH and HIS in three different strains, the fermentation productions-NMN SAM and VD13 exhibited a remarkable enhancement of 30 %–70 %. The findings presented here uncovered the terminator characteristics contributed to the upstream mRNA stability, providing guiding principles for gene circuit design.
{"title":"Regulatory significance of terminator: A systematic approach for dissecting terminator-mediated enhancement of upstream mRNA stability","authors":"Jia-Wei Ren , Jin-Peng Zhang , Zi-Lun Mei , Jia-Yi Shao , Guo-Qiang Xu , Hui Li , Jin-Song Gong , Xiao-Mei Zhang , Jin-Song Shi , Xiao-juan Zhang , Zheng-hong Xu","doi":"10.1016/j.synbio.2024.11.006","DOIUrl":"10.1016/j.synbio.2024.11.006","url":null,"abstract":"<div><div>The primary function of terminators is to terminate transcription in gene expression. Although some studies have suggested that terminators also contribute positively to upstream gene expression, the extent and underlying mechanism of this effect remain largely unexplored. Here, the correlation between terminating strength and upstream mRNA stability was investigated by constructing a terminator mutation library through randomizing 5 nucleotides, assisted by FlowSeq technology, terminator variants were categorized based on the downstream fluorescence intensity, followed by high-throughput sequencing. To examine the impact of terminators on mRNA stability, the abundance of downstream gene transcripts for each terminator variant was quantified through cDNA sequencing. The results revealed that the transcript abundance controlled by strong terminators was, on average 2.2 times greater than those controlled by weak terminators on average. Moreover, several distinct features could be ascribed to high relative abundance of upstream gene transcript, including a high GC content at the base region of hairpin, and a high AT content in downstream of the U-tract. Additionally, these terminators showed a free energy between −28 and −22 kcal/mol, and a stem length of 14 nt. Finally, these features ascribed the upstream beneficial terminator were validated across various expression systems. By incorporating the optimal terminator downstream of RSF, GSH and HIS in three different strains, the fermentation productions-NMN SAM and VD13 exhibited a remarkable enhancement of 30 %–70 %. The findings presented here uncovered the terminator characteristics contributed to the upstream mRNA stability, providing guiding principles for gene circuit design.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 326-335"},"PeriodicalIF":4.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11696848/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142932451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-31DOI: 10.1016/j.synbio.2024.12.009
Xin Ni , Jingjing Li , Wei Yu , Fan Bai , Zongbao K. Zhao , Jiaoqi Gao , Fan Yang , Yongjin J. Zhou
Lignocellulose bio-refinery via microbial cell factories for chemical production represents a renewable and sustainable route in response to resource starvation and environmental concerns. However, the challenges associated with the co-utilization of xylose and glucose often hinders the efficiency of lignocellulose bioconversion. Here, we engineered yeast Ogataea polymorpha to effectively produce free fatty acids from lignocellulose. The non-oxidative branch of the pentose phosphate pathway, and the adaptive expression levels of xylose metabolic pathway genes XYL1, XYL2 and XYL3, were systematically optimized. In addition, the introduction of xylose transporter and global regulation of transcription factors achieved synchronous co-utilization of glucose and xylose. The engineered strain produced 11.2 g/L FFAs from lignocellulose hydrolysates, with a yield of up to 0.054 g/g. This study demonstrated that metabolic rewiring of xylose metabolism could support the efficient co-utilization of glucose and xylose from lignocellulosic resources, which may provide theoretical reference for lignocellulose biorefinery.
{"title":"High-level production of free fatty acids from lignocellulose hydrolysate by co-utilizing glucose and xylose in yeast","authors":"Xin Ni , Jingjing Li , Wei Yu , Fan Bai , Zongbao K. Zhao , Jiaoqi Gao , Fan Yang , Yongjin J. Zhou","doi":"10.1016/j.synbio.2024.12.009","DOIUrl":"10.1016/j.synbio.2024.12.009","url":null,"abstract":"<div><div>Lignocellulose bio-refinery via microbial cell factories for chemical production represents a renewable and sustainable route in response to resource starvation and environmental concerns. However, the challenges associated with the co-utilization of xylose and glucose often hinders the efficiency of lignocellulose bioconversion. Here, we engineered yeast <em>Ogataea polymorpha</em> to effectively produce free fatty acids from lignocellulose. The non-oxidative branch of the pentose phosphate pathway, and the adaptive expression levels of xylose metabolic pathway genes <em>XYL1</em>, <em>XYL2</em> and <em>XYL3</em>, were systematically optimized. In addition, the introduction of xylose transporter and global regulation of transcription factors achieved synchronous co-utilization of glucose and xylose. The engineered strain produced 11.2 g/L FFAs from lignocellulose hydrolysates, with a yield of up to 0.054 g/g. This study demonstrated that metabolic rewiring of xylose metabolism could support the efficient co-utilization of glucose and xylose from lignocellulosic resources, which may provide theoretical reference for lignocellulose biorefinery.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 401-409"},"PeriodicalIF":4.4,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758827/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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