ACS Synthetic Biology最新文献_第5页

Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast 二酰基甘油酰基转移酶底物结合袋的突变改变酵母中三酰基甘油酯的脂肪酸组成。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-03 DOI: 10.1021/acssynbio.5c00143

Nazreen V. M. Abdul Muthaliff, Nur Eka Fitriani, Derek Smith, Jing Sen Ong, Lay Kien Yang, Coleen Toledo Busran, Aaron Thong, Prakash Arumugam and Naazneen Sofeo*,

{"title":"Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast","authors":"Nazreen V. M. Abdul Muthaliff, Nur Eka Fitriani, Derek Smith, Jing Sen Ong, Lay Kien Yang, Coleen Toledo Busran, Aaron Thong, Prakash Arumugam and Naazneen Sofeo*, ","doi":"10.1021/acssynbio.5c00143","DOIUrl":"10.1021/acssynbio.5c00143","url":null,"abstract":"Triacylglycerols (TAGs) are the main components of food oils and fats. The fatty acid composition of TAGs varies for different oils and fats. Specific enzymes sequentially add three fatty acids to the glycerol backbone of TAGs. Diacylglycerol acyltransferase or DGAT adds the third and ultimate fatty acid to the glycerol backbone at the sn-3 position. In this study, we characterized the substrate-binding pocket of enzyme DGAT1 from Arabidopsis thaliana through heterologous expression in the DGAT mutant of Saccharomyces cerevisiae. We performed site saturation mutagenesis on 10 amino acid residues in the catalytic site and examined their effects on the fatty acid profile of yeast cells. Our results indicate that mutations F373G, T240I, M289F, and V248I impact the yeast TAG profile either in terms of the total saturation level or the carbon chain length of the fatty acids, suggesting that they change the DGAT’s substrate preference. This offers insights into crucial amino acid residues in the DGAT binding pocket which can be engineered for fine tuning the lipid profile. In summary, we have harnessed the power of enzyme engineering to modify the fatty acyl makeup of triglycerides and created a sustainable platform for the production of customized alternative lipids.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3401–3413"},"PeriodicalIF":3.9,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Diversifying Substrates and Reaction Conditions for Polymerase Strand Recycling 聚合酶链再循环的多样化底物和反应条件。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-03 DOI: 10.1021/acssynbio.5c00207

Yueyi Li, Arno Gundlach, Andrew Ellington and Julius B. Lucks*,

引用次数: 0

Catalytic Residue Reprogramming Enhances Enzyme Activity at Alkaline pH via Phenolate-Mediated Proton Transfer 催化残基重编程通过苯酚介导的质子转移增强酶活性。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-02 DOI: 10.1021/acssynbio.5c00379

Peerapak Vajanapanich, Parinthon Nearmnala, Jinjutha Parkbhorn, Bodee Nutho, Thanyada Rungrotmongkol and Narupat Hongdilokkul*,

{"title":"Catalytic Residue Reprogramming Enhances Enzyme Activity at Alkaline pH via Phenolate-Mediated Proton Transfer","authors":"Peerapak Vajanapanich, Parinthon Nearmnala, Jinjutha Parkbhorn, Bodee Nutho, Thanyada Rungrotmongkol and Narupat Hongdilokkul*, ","doi":"10.1021/acssynbio.5c00379","DOIUrl":"10.1021/acssynbio.5c00379","url":null,"abstract":"Achieving efficient enzyme catalysis under extreme pH conditions remains a major challenge in biocatalysis and synthetic biology. To address this, we present an enzyme engineering strategy that integrates rational redesign of catalytic residues with directed evolution to enable robust enzyme function at alkaline pH. The core principle involves replacing the conserved general base with an ionizable residue of higher intrinsic pKa, shifting the proton transfer mechanism from carboxylate- to phenolate-mediated catalysis. Previously, we engineered TEM β-lactamase by substituting the universally conserved Glu166 with tyrosine (E166Y), which severely impaired activity. Directed evolution subsequently restored function, yielding the optimized variant YR5-2. Although this engineering effort originally aimed to validate a novel selection platform, the evolutionary trajectory of YR5-2 exemplifies our proposed strategy in the present study. Here, we characterize YR5-2 and its parental variants across a wide pH range. Steady-state kinetic analyses reveal a > 3-unit shift in the optimal pH for kcat, with YR5-2 reaching 870 s–1 at pH 10.0, a kcat value comparable to that of the wild type at its optimal pH. Kinetic analyses of Y166E revertants, together with molecular dynamics simulations, support a mechanistic transition in which Tyr166 functions as the catalytic general base. In vivo experiments further demonstrate the utility of YR5-2 as a selectable marker by enabling recombinant protein expression in E. coli under alkaline growth conditions. This work establishes a broadly applicable framework for reprogramming enzyme catalytic mechanisms, particularly in hydrolases, to expand their operational pH range and unlock new opportunities in industrial and environmental biocatalysis.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3612–3623"},"PeriodicalIF":3.9,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5c00379","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935722","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}

引用次数: 0

Complex Kinetic Models Predict β-Carotene Production and Reveal Flux Limitations in Recombinant Saccharomyces cerevisiae Strains 复杂动力学模型预测重组酿酒酵母菌株β-胡萝卜素产量并揭示通量限制。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-02 DOI: 10.1021/acssynbio.5c00256

Benjamín R. Elizondo, and , Pedro A. Saa*,

{"title":"Complex Kinetic Models Predict β-Carotene Production and Reveal Flux Limitations in Recombinant Saccharomyces cerevisiae Strains","authors":"Benjamín R. Elizondo,  and , Pedro A. Saa*, ","doi":"10.1021/acssynbio.5c00256","DOIUrl":"10.1021/acssynbio.5c00256","url":null,"abstract":"β-Carotene is a high-value compound with multiple commercial applications as a pigment and due to its antioxidant properties. For its industrial production, precision fermentation using engineered microorganisms has been proposed as an attractive alternative given consumer concerns and technical limitations of traditional production methods such as chemical synthesis and extraction from plants. However, the factors limiting microbial production are complex and remain poorly understood, hindering bioprocess scale-up. To tackle this limitation, we built and evaluated kinetic model ensembles of the native mevalonate and the heterologous β-carotene production pathways in recombinant Saccharomyces cerevisiae strains to identify bottlenecks limiting the production flux. For this task, flux and transcriptomic data from chemostat cultivations were generated and combined with literature information for simulating model structures capturing different degrees of kinetic detail and complexity within the ABC-GRASP framework. Our results showed that detailed kinetic models including both allosteric regulation and complex mechanistic descriptions (e.g., enzyme promiscuity) are necessary to explain the metabolic phenotype of recombinant strains in different conditions. Calculation of flux and concentration response coefficients of the detailed models revealed that the promiscuous CrtYB enzyme exerts the highest control over β-carotene production at different growth rates in the best producer. Simulation of various enzyme and metabolite perturbations confirmed the above result and discarded other seemingly intuitive targets for intervention, e.g., upregulation of ERG10. Overall, this work deepens our understanding about the factors limiting β-carotene production in yeast, providing mechanistic models for in silico metabolic prospection and rational design of genetic interventions.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3457–3472"},"PeriodicalIF":3.9,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5c00256","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935710","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}

引用次数: 0

Multichassis Expression of Cyanobacterial and Other Bacterial Biosynthetic Gene Clusters 蓝藻和其他细菌生物合成基因簇的多底盘表达。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-02 DOI: 10.1021/acssynbio.5c00390

Dipesh Dhakal, Campbell W. Eckhardt, Yujia Jiang, Hendrik Luesch and Yousong Ding*,

{"title":"Multichassis Expression of Cyanobacterial and Other Bacterial Biosynthetic Gene Clusters","authors":"Dipesh Dhakal, Campbell W. Eckhardt, Yujia Jiang, Hendrik Luesch and Yousong Ding*, ","doi":"10.1021/acssynbio.5c00390","DOIUrl":"10.1021/acssynbio.5c00390","url":null,"abstract":"Heterologous expression of biosynthetic gene clusters (BGCs) is a powerful strategy for natural product (NP) discovery, yet achieving consistent expression across microbial hosts remains challenging. Here, we developed cross-phyla vector systems enabling the expression of BGCs from cyanobacteria and other bacterial origins in Gram-negative Escherichia coli, Gram-positive Bacillus subtilis, and two model cyanobacterial strains including unicellular Synechocystis PCC 6803 and filamentous Anabaena sp. PCC 7120. Following validation using constitutive and inducible expression of the enhanced yellow fluorescent protein (eYFP), we applied these vectors to express the shinorine and violacein BGCs in all four hosts. Promoter tuning, substrate feeding, BGC refactoring, and inducible control enhanced NP production and mitigated host toxicity. Notably, we demonstrated that B. subtilis can serve as a chassis for cyanobacterial NP BGC expression. Our results provide versatile expression platforms for probing BGC function and accelerating natural product discovery from diverse cyanobacterial and other bacterial lineages.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3356–3361"},"PeriodicalIF":3.9,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

APEX: Automated Protein EXpression in Escherichia coli APEX：在大肠杆菌中自动蛋白表达。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-02 DOI: 10.1021/acssynbio.5c00189

Martyna Kasprzyk, Michael A. Herrera and Giovanni Stracquadanio*,

{"title":"APEX: Automated Protein EXpression in Escherichia coli","authors":"Martyna Kasprzyk, Michael A. Herrera and Giovanni Stracquadanio*, ","doi":"10.1021/acssynbio.5c00189","DOIUrl":"10.1021/acssynbio.5c00189","url":null,"abstract":"Heterologous protein expression is an indispensable strategy for generating recombinant proteins. Escherichia coli (E. coli) is the most widely used microbial host for recombinant protein production due to its rapid growth, well-characterized genetics, and ability to produce recombinant proteins in high yields using modern recombinant DNA technology. However, while there is a plethora of robust protein expression protocols for E. coli, these methods are often unsuitable for high-throughput screening due to their significant resource and time consumption; these protocols are also susceptible to operator error and inconsistency. To address these challenges, we developed Automated Protein EXpression (APEX), a robust and automated pipeline for recombinant protein production in E. coli. APEX leverages the accessible, open-source Opentrons OT-2 platform to automate microbial handling and protein expression with high precision and reproducibility. APEX can be configured to perform heat shock transformation, selective plating, colony sampling, microculturing, and protein expression induction using a low-cost, minimal OT-2 hardware setup. We further demonstrate the efficacy of our automated transformation workflows using a variety of plasmids (2.7–17.7 kb) and exemplify the automated heterologous expression of a diverse array of proteins (27–222 kDa). Designed with customization, modularity, and user-friendliness in mind, APEX can be easily adapted to the operator’s needs without requiring any coding expertise. APEX is available at https://github.com/stracquadaniolab/apex-nf under the AGPL3 license.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3434–3444"},"PeriodicalIF":3.9,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor 利用CRISPR/ cas9碱基编辑器增强环形红酵母游离脂肪酸的生成

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-01 DOI: 10.1021/acssynbio.5c00359

Yi Yu, Quan Yuan, Zihe Liu, Baisong Tong and Shuobo Shi*,

{"title":"Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor","authors":"Yi Yu, Quan Yuan, Zihe Liu, Baisong Tong and Shuobo Shi*, ","doi":"10.1021/acssynbio.5c00359","DOIUrl":"10.1021/acssynbio.5c00359","url":null,"abstract":"Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3578–3588"},"PeriodicalIF":3.9,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Isolation of Synechocystis Mutants Overproducing Mannitol Directly from CO2 via Laboratory Evolution under Increasing Salt Concentration 增加盐浓度下实验室进化从CO2中分离过量生产甘露醇的聚囊菌突变体。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-01 DOI: 10.1021/acssynbio.5c00344

Wenyang Wu, Jente A. Jongbloets, Wei Du, Klaas J. Hellingwerf and Filipe Branco dos Santos*,

{"title":"Isolation of Synechocystis Mutants Overproducing Mannitol Directly from CO2 via Laboratory Evolution under Increasing Salt Concentration","authors":"Wenyang Wu, Jente A. Jongbloets, Wei Du, Klaas J. Hellingwerf and Filipe Branco dos Santos*, ","doi":"10.1021/acssynbio.5c00344","DOIUrl":"10.1021/acssynbio.5c00344","url":null,"abstract":"Mannitol is a naturally occurring C(6) polyol with a wide range of applications in the food and pharmaceutical industry. In a previous study, mannitol production was achieved via the direct conversion of CO2 in Synechocystis sp. PCC6803. However, a major barrier to future applications of this strain was its low production rate. In this study, three mutants were isolated after 164 generations of adaptive laboratory evolution under salt stress. These mannitol overproducing mutants were able to produce 27.71 mg L–1 OD730–1 mannitol under 350 mM salt stress when the OD730 reached 2, roughly 24 times higher than their parental strains. Whole-genome resequencing was then performed, revealing mutations in 2 genes─pnp and sigA/rpoD1─of the overproducing mutants when compared to the parental strain. Of these genes, pnp which encodes for polyribonucleotide nucleotidyltransferase was found to negatively affect mannitol production in cells via reverse engineering methods, in which the (partial) removal of pnp alone resulted in a 6.4-fold increase in the mannitol production. The work reported here substantially advances mannitol production capabilities in engineered Synechocystis sp. PCC6803 strains through adaptive evolution but also highlights the previously unrecognized negative regulatory role of pnp in mannitol synthesis.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3557–3567"},"PeriodicalIF":3.9,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5c00344","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935840","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}

引用次数: 0

Enhanced Menaquinone Biosynthesis by Engineering 2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate Synthase MenD to Alleviate Feedback Inhibition in Bacillus subtilis 工程修饰2-琥珀酰-5-烯醇丙酮酰-6-羟基-3-环己二烯-1-羧酸合酶MenD促进甲基萘醌生物合成以减轻枯草芽孢杆菌的反馈抑制

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-08-28 DOI: 10.1021/acssynbio.5c00419

Xian Sun, Yangyang Li, Lei Xing, Siqi Liu, Xianhao Xu, Yanfeng Liu, Jianghua Li, Jian Chen, Xueqin Lv, Hua Yin* and Long Liu*,

{"title":"Enhanced Menaquinone Biosynthesis by Engineering 2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate Synthase MenD to Alleviate Feedback Inhibition in Bacillus subtilis","authors":"Xian Sun, Yangyang Li, Lei Xing, Siqi Liu, Xianhao Xu, Yanfeng Liu, Jianghua Li, Jian Chen, Xueqin Lv, Hua Yin* and Long Liu*, ","doi":"10.1021/acssynbio.5c00419","DOIUrl":"10.1021/acssynbio.5c00419","url":null,"abstract":"Vitamin K2 (menaquinones, MKs), a group of fat-soluble vitamins with a 2-methyl-1,4-naphthoquinone core, plays vital roles in bone metabolism and cardiovascular health. Although microbial biosynthesis has become the primary method for MKs production, regulatory constraints and metabolic bottlenecks in the MK pathway limit its production efficiency. In this study, we identified that 1,4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the MK pathway of Bacillus subtilis, exerts feedback inhibition on the key enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC) synthase MenD. Through structure-guided evolution, we obtained a feedback-resistant variant (MenDW322I/R323F) with 43% higher activity than wild-type, maintaining full function at 1 μM DHNA. This mutant boosted Menaquinone-7 (MK-7) production by 22.3% to 186.5 mg/L. Molecular dynamics (MD) simulation indicates that the attenuation of the feedback inhibition results from weakened interaction forces between the mutant and DHNA. This work advances the understanding of MKs pathway regulation and paves the way for high-efficiency microbial production of MKs.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3709–3720"},"PeriodicalIF":3.9,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhancing Astaxanthin Biosynthesis in Synthetic Yeast Through Combinatorial Metabolic Engineering and Genome-Scale Evolution 通过组合代谢工程和基因组尺度进化促进合成酵母虾青素的生物合成。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-08-28 DOI: 10.1021/acssynbio.5c00376

Shuilian Guo, Shangfeng Liao, Haixin Chen, Chuang Xue, Huiming Zhang, Zhen Yue, Xiuqing Zhang and Xiaodong Fang*,

{"title":"Enhancing Astaxanthin Biosynthesis in Synthetic Yeast Through Combinatorial Metabolic Engineering and Genome-Scale Evolution","authors":"Shuilian Guo, Shangfeng Liao, Haixin Chen, Chuang Xue, Huiming Zhang, Zhen Yue, Xiuqing Zhang and Xiaodong Fang*, ","doi":"10.1021/acssynbio.5c00376","DOIUrl":"10.1021/acssynbio.5c00376","url":null,"abstract":"Astaxanthin, a high-value keto-carotenoid with exceptional antioxidant capacity, has significant commercial potential for industrial applications. Microbial biosynthesis via engineered synthetic yeast presents an environmentally sustainable production platform. In this study, we developed a multistrategy optimization framework to enhance astaxanthin biosynthesis in synthetic yeast. Our systematic approach initiated with the construction of a de novo astaxanthin pathway in synthetic yeast strain 2369R, achieving a baseline production of 0.11 mg/L. Through rigorous screening of heterologous enzymes, we identified optimal variants of β-carotene hydroxylase (CrtZ) and ketolase (CrtW) that increased the titer to 0.65 mg/L. Subsequently, the combined enhancement of MVA pathway flux (via tHMG1 overexpression) and lipid metabolism regulation (through DGK1 overexpression) synergistically boosted astaxanthin production to 2.59 mg/L. Through combinatorial implementation of genome-scale diversification using the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system coupled with an absorption-based semi-high-throughput screening platform (A450/A600), we successfully isolated an elite mutant strain, YgM97, that achieved 6.85 mg/L astaxanthin production in shake-flask culture. This represents a remarkable 61.27-fold enhancement compared with the parental strain. Transcriptomic and genomic analyses subsequently revealed the potential molecular mechanisms underlying this significant yield improvement. Collectively, this study demonstrates the powerful synergy between rational metabolic engineering and randomized genome evolution, providing a novel paradigm for high-value compound biosynthesis in a microbial chassis.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3589–3599"},"PeriodicalIF":3.9,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0