Pub Date : 2026-03-16DOI: 10.1016/j.ymben.2026.03.009
Michael J. Ream, Kristala L.J. Prather
Engineers have effectively employed quorum sensing (QS) in a variety of applications to dynamically regulate gene expression. Particular emphasis has been placed on the class of well-studied systems that use acyl homoserine lactones (AHL) as signaling molecules due to their ease of implementation, high expression level, and previous optimization efforts. However, many of these AHL systems respond to ligands with similar structures, causing crosstalk when combined in multi-layered regulation strategies. Here, we first confirmed the functional orthogonality of the previously identified Tra and Rpa quorum sensing circuits within a single strain of Escherichia coli MG1655 by analyzing the pairwise interactions of several AHL systems. The orthogonality of the systems allowed for independent tuning of two control strategies, which was then applied to the naringenin biosynthetic pathway. The Tra system was used to activate expression of tyrosine-ammonia lyase (TAL) and 4-coumaroyl-CoA ligase (4CL), controlling the expression of the upstream pathway. Meanwhile, Rpa dynamically downregulated competing pathways of native metabolism via CRISPRi to increase availability of malonyl-CoA. This multi-layered approach provided finely-tuned metabolic control that allowed for a combinatorial screening of optimal dynamic regulation. A strain library with varying promoter strengths was then built to test AHL induction timings and screened for target compound production. Naringenin production from this autoinducible method reached a final titer of 71.02 ± 3.96 mg/L in flask-scale fermentation.
{"title":"Orthogonal quorum sensing circuits enable dynamic regulation in Escherichia coli","authors":"Michael J. Ream, Kristala L.J. Prather","doi":"10.1016/j.ymben.2026.03.009","DOIUrl":"https://doi.org/10.1016/j.ymben.2026.03.009","url":null,"abstract":"Engineers have effectively employed quorum sensing (QS) in a variety of applications to dynamically regulate gene expression. Particular emphasis has been placed on the class of well-studied systems that use acyl homoserine lactones (AHL) as signaling molecules due to their ease of implementation, high expression level, and previous optimization efforts. However, many of these AHL systems respond to ligands with similar structures, causing crosstalk when combined in multi-layered regulation strategies. Here, we first confirmed the functional orthogonality of the previously identified Tra and Rpa quorum sensing circuits within a single strain of <ce:italic>Escherichia coli</ce:italic> MG1655 by analyzing the pairwise interactions of several AHL systems. The orthogonality of the systems allowed for independent tuning of two control strategies, which was then applied to the naringenin biosynthetic pathway. The Tra system was used to activate expression of tyrosine-ammonia lyase (TAL) and 4-coumaroyl-CoA ligase (4CL), controlling the expression of the upstream pathway. Meanwhile, Rpa dynamically downregulated competing pathways of native metabolism via CRISPRi to increase availability of malonyl-CoA. This multi-layered approach provided finely-tuned metabolic control that allowed for a combinatorial screening of optimal dynamic regulation. A strain library with varying promoter strengths was then built to test AHL induction timings and screened for target compound production. Naringenin production from this autoinducible method reached a final titer of 71.02 ± 3.96 mg/L in flask-scale fermentation.","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"49 1","pages":""},"PeriodicalIF":8.4,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147465875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-13DOI: 10.1016/j.ymben.2026.03.004
Guido Zampieri, Viktor Sandner, Suraj Verma, Julia Kraemer, Christopher Lennon, Annalisa Occhipinti, Graham McCreath, Claudio Angione
{"title":"Bioprocess optimisation via joint machine learning and metabolic modelling","authors":"Guido Zampieri, Viktor Sandner, Suraj Verma, Julia Kraemer, Christopher Lennon, Annalisa Occhipinti, Graham McCreath, Claudio Angione","doi":"10.1016/j.ymben.2026.03.004","DOIUrl":"https://doi.org/10.1016/j.ymben.2026.03.004","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"1 1","pages":""},"PeriodicalIF":8.4,"publicationDate":"2026-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147447612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-19DOI: 10.1016/j.ymben.2025.11.015
Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu
Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in Y. lipolytica. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.
{"title":"Engineering amino acid-derived malonyl-CoA pathways to boost polyketide production in Yarrowia lipolytica","authors":"Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu","doi":"10.1016/j.ymben.2025.11.015","DOIUrl":"10.1016/j.ymben.2025.11.015","url":null,"abstract":"<div><div>Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in <em>Y. lipolytica</em>. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 99-109"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145553880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-05DOI: 10.1016/j.ymben.2025.11.004
Juthamas Jaroensuk , Joshua P. Abraham , Baltazar E. Zuniga , Hawkins S. Shepard , Michael Wei , Russell Williams , Stewart A. Morley , Maneesh Lingwan , Jiahong Zhou , Michael A. Jindra , Poonam Jyoti , Bo Wang , Jody C. May , John A. McLean , Jamey D. Young , Brian F. Pfleger , Doug K. Allen
Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In Escherichia coli, which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, Picosynechococcus sp. PCC 7002 and the Δaas strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δaas strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.
{"title":"Disruption of acyl-acyl carrier protein (acyl-ACP) synthetase in cyanobacteria impairs lipid remodeling as revealed by acyl-ACP measurements","authors":"Juthamas Jaroensuk , Joshua P. Abraham , Baltazar E. Zuniga , Hawkins S. Shepard , Michael Wei , Russell Williams , Stewart A. Morley , Maneesh Lingwan , Jiahong Zhou , Michael A. Jindra , Poonam Jyoti , Bo Wang , Jody C. May , John A. McLean , Jamey D. Young , Brian F. Pfleger , Doug K. Allen","doi":"10.1016/j.ymben.2025.11.004","DOIUrl":"10.1016/j.ymben.2025.11.004","url":null,"abstract":"<div><div>Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In <em>Escherichia coli,</em> which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, <em>Picosynechococcus</em> sp. PCC 7002 and the Δ<em>aas</em> strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δ<em>aas</em> strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 45-56"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-10DOI: 10.1016/j.ymben.2025.11.009
Sami Kazaz , Yu-Ton Chen , Senri Yamamoto , Yang Tian , Chien-Yuan Lin , Dylan Chin , İrem Pamukçu , Ibraheem Mohammed Al Shammaa , Yusuf Selman Akbas , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Albert P. Kausch , Yuki Tobimatsu , Aymerick Eudes
The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.
{"title":"Engineered plants for the production of the antioxidants arbutin and gallate","authors":"Sami Kazaz , Yu-Ton Chen , Senri Yamamoto , Yang Tian , Chien-Yuan Lin , Dylan Chin , İrem Pamukçu , Ibraheem Mohammed Al Shammaa , Yusuf Selman Akbas , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Albert P. Kausch , Yuki Tobimatsu , Aymerick Eudes","doi":"10.1016/j.ymben.2025.11.009","DOIUrl":"10.1016/j.ymben.2025.11.009","url":null,"abstract":"<div><div>The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in <em>Nicotiana benthamiana</em> leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from <em>Pseudomonas</em> sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from <em>Candida parapsilosis</em>. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale <em>in planta</em> production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 57-66"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-11DOI: 10.1016/j.ymben.2025.12.001
Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi
Human milk oligosaccharides (HMOs), such as lacto-N-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in Escherichia coli that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-N-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.
{"title":"Lacto-N-tetraose biosynthesis from lactose via metabolically rewired Escherichia coli","authors":"Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi","doi":"10.1016/j.ymben.2025.12.001","DOIUrl":"10.1016/j.ymben.2025.12.001","url":null,"abstract":"<div><div>Human milk oligosaccharides (HMOs), such as lacto-<em>N</em>-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in <em>Escherichia coli</em> that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-<em>N</em>-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 182-191"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-16DOI: 10.1016/j.ymben.2025.12.005
Mengyi Xiong , Zhiqiang Du , Zehao Fan , Beibei Wang , Wenjiao Diao , Min Wang , Xuenian Huang , Xuefeng Lu
Pravastatin is a widely prescribed cholesterol-lowering drug known for its superior water solubility and favorable pharmacokinetics. However, its industrial production remains constrained by an inefficient two-step fermentation process, particularly the second biotransformation step involving Streptomyces fermentation. In this study, we engineered the industrial mevastatin-producing strain Penicillium citrinum MEFC10 to achieve efficient one-step pravastatin biosynthesis. Through systematic screening and integration of optimal cytochrome P450-redox partner modules, a one-step pravastatin production cell factory was constructed in industrial Penicillium citrinum MEFC10. Next, NADP+-dependent g6pd3 was overexpressed to increase statin biosynthesis via NADPH regeneration. Further manipulation of pathway transcriptional regulator, self-resistance gene and minimization of byproduct formation, a high-performance Pra2.0 strain was constructed. The Pra2.0 strain produced 8.48 g/L pravastatin and 15.06 g/L total statins in a 50-L bioreactor under fed-batch fermentation. This work established a one-step fermentation process for pravastatin production with markedly improved efficiency over the conventional methods. This work not only establishes an efficient, green production route for pravastatin but also provides a versatile engineering framework for the sustainable biosynthesis of other complex fungal polyketides.
{"title":"Systematic metabolic engineering of an industrial Penicillium citrinum for one-step pravastatin production","authors":"Mengyi Xiong , Zhiqiang Du , Zehao Fan , Beibei Wang , Wenjiao Diao , Min Wang , Xuenian Huang , Xuefeng Lu","doi":"10.1016/j.ymben.2025.12.005","DOIUrl":"10.1016/j.ymben.2025.12.005","url":null,"abstract":"<div><div>Pravastatin is a widely prescribed cholesterol-lowering drug known for its superior water solubility and favorable pharmacokinetics. However, its industrial production remains constrained by an inefficient two-step fermentation process, particularly the second biotransformation step involving <em>Streptomyces fermentation</em>. In this study, we engineered the industrial mevastatin-producing strain <em>Penicillium citrinum</em> MEFC10 to achieve efficient one-step pravastatin biosynthesis. Through systematic screening and integration of optimal cytochrome P450-redox partner modules, a one-step pravastatin production cell factory was constructed in industrial <em>Penicillium citrinum</em> MEFC10. Next, NADP<sup>+</sup>-dependent <em>g6pd3</em> was overexpressed to increase statin biosynthesis via NADPH regeneration. Further manipulation of pathway transcriptional regulator, self-resistance gene and minimization of byproduct formation, a high-performance Pra2.0 strain was constructed. The Pra2.0 strain produced 8.48 g/L pravastatin and 15.06 g/L total statins in a 50-L bioreactor under fed-batch fermentation. This work established a one-step fermentation process for pravastatin production with markedly improved efficiency over the conventional methods. This work not only establishes an efficient, green production route for pravastatin but also provides a versatile engineering framework for the sustainable biosynthesis of other complex fungal polyketides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 223-230"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145777265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-17DOI: 10.1016/j.ymben.2026.01.008
Bruno S. Paulo, Sean B. Romanowski, Adjo E. Kadjo, Vitor B. Lourenzon, Alessandra S. Eustáquio
Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing Burkholderia sp. FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20–40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2− strain produced only 5–23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1− p2− strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.
{"title":"Genome minimization of a Burkholderia bacterial host","authors":"Bruno S. Paulo, Sean B. Romanowski, Adjo E. Kadjo, Vitor B. Lourenzon, Alessandra S. Eustáquio","doi":"10.1016/j.ymben.2026.01.008","DOIUrl":"10.1016/j.ymben.2026.01.008","url":null,"abstract":"<div><div>Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing <em>Burkholderia sp.</em> FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20–40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2<sup>−</sup> strain produced only 5–23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1<sup>−</sup> p2<sup>−</sup> strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 305-314"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145995161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-10DOI: 10.1016/j.ymben.2026.01.004
Yunjie Li , Yujiao Lu , Pingping Han , Qingqing Guo , Shuo Wang , Zhongyi Jiang , Yi-Heng P. Job Zhang
The biosynthesis of polysaccharides with precisely defined structures, such as tunable degree of polymerization (DP) and low polydispersity index (PDI), remains a significant challenge in microbial cell factories due to their intricate endogenous metabolic networks. In vitro metabolic engineering (ivME) has emerged as a promising alternative, offering simplified pathway design and easy process optimization. In this study, ivME was utilized for the precise synthesis of β-1,2-glucans from β-1,4-linked cellobiose. A four-enzyme system comprising cellobiose phosphorylase, β-1,2-oligoglucan phosphorylase, glucose oxidase, and catalase operated under pH self-neutralized conditions, efficiently producing β-1,2-glucan at a concentration of 31.9 ± 0.4 g/L with a high molar yield (93.3 ± 1.3 %) and a rapid productivity of 4.0 ± 0.1 g/L/h. β-1,2-Glucans with tunable DPs (75–531) and narrow molecular weight distributions (PDI as low as 1.2) were synthesized by adjusting primer concentration, enzyme loadings, and reaction time. The low PDI values of β-1,2-glucans were attributed to the smart pathway design, the careful selection of β-1,2-oligoglucan phosphorylase, and the use of sophorose as the primer. The DP values were mainly influenced by the concentration and type of primers with sophorose outperforming glucose. This strategy of direct glycosidic bond rearrangement from β-1,4 to β-1,2 linkages without coenzymes (e.g., CoA, NAD, ATP, UTP) or external energy input provided a new route for lignocellulosic biomass utilization and significantly enhanced the capabilities of ivME for the production of tailored polysaccharides.
{"title":"Precise biosynthesis of β-1,2-glucan from cellulosic materials by in vitro metabolic engineering","authors":"Yunjie Li , Yujiao Lu , Pingping Han , Qingqing Guo , Shuo Wang , Zhongyi Jiang , Yi-Heng P. Job Zhang","doi":"10.1016/j.ymben.2026.01.004","DOIUrl":"10.1016/j.ymben.2026.01.004","url":null,"abstract":"<div><div>The biosynthesis of polysaccharides with precisely defined structures, such as tunable degree of polymerization (DP) and low polydispersity index (PDI), remains a significant challenge in microbial cell factories due to their intricate endogenous metabolic networks. <em>In vitro</em> metabolic engineering (<em>iv</em>ME) has emerged as a promising alternative, offering simplified pathway design and easy process optimization. In this study, <em>iv</em>ME was utilized for the precise synthesis of β-1,2-glucans from β-1,4-linked cellobiose. A four-enzyme system comprising cellobiose phosphorylase, β-1,2-oligoglucan phosphorylase, glucose oxidase, and catalase operated under pH self-neutralized conditions, efficiently producing β-1,2-glucan at a concentration of 31.9 ± 0.4 g/L with a high molar yield (93.3 ± 1.3 %) and a rapid productivity of 4.0 ± 0.1 g/L/h. β-1,2-Glucans with tunable DPs (75–531) and narrow molecular weight distributions (PDI as low as 1.2) were synthesized by adjusting primer concentration, enzyme loadings, and reaction time. The low PDI values of β-1,2-glucans were attributed to the smart pathway design, the careful selection of β-1,2-oligoglucan phosphorylase, and the use of sophorose as the primer. The DP values were mainly influenced by the concentration and type of primers with sophorose outperforming glucose. This strategy of direct glycosidic bond rearrangement from β-1,4 to β-1,2 linkages without coenzymes (e.g., CoA, NAD, ATP, UTP) or external energy input provided a new route for lignocellulosic biomass utilization and significantly enhanced the capabilities of <em>iv</em>ME for the production of tailored polysaccharides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 284-294"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145955932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2026-01-14DOI: 10.1016/j.ymben.2026.01.005
Sonali Srivastava , Aakash Chandramouli , Payal Gupta , Abdur Rahman Manzer , Rahul Choudhury , D. Srinivasa Reddy , Syed Shams Yazdani , Siddhesh S. Kamat , Debasisa Mohanty , Vinay K. Nandicoori , Rajesh S. Gokhale
Delta lactones are fatty acid-derived aroma compounds that hold tremendous commercial value. As consumer demand for natural flavours continues to rise, the bioproduction of δ-lactones, including δ-decalactone and δ-dodecalactone, is attracting substantial interest. Our study brings forth a novel approach to the bioproduction of δ-lactones from glucose, deviating from existing methods that primarily rely on the biotransformation of fatty acids. The high cost of fatty acid raw material constrains the commercial viability of this traditional approach. We engineered surface-lipid producing type I polyketide synthase (PKS) from Mycobacterium smegmatis by incorporating macrolactone thioesterase (TE) domain. Two out of three fusion constructs produced an appropriately engineered PKS-TE fusion protein that facilitated the synthesis of δ-lactones. When grown on glucose as the sole carbon source, recombinant E. coli expressing the engineered PKS-TE fusion protein successfully made δ-lactones ranging from C8-C18 acyl chains. Our research further highlights the potential of Mycobacterium smegmatis as a cell factory for producing fatty acid-based δ-lactones. By genetically designing and engineering Mycobacterium smegmatis to express PKS-TE fusion protein, we achieved bioproduction of various δ-lactones. Batch fermentation of the engineered E. coli strain fed with 2 % glucose produced 786 mg/L of δ-dodecalactone and 444 mg/L of δ-decalactone at 120 h, underscoring the efficacy of our approach. Thus, this study is the first to demonstrate a methodology for redirecting primary metabolic intermediates towards δ-lactone biosynthesis in engineered bacteria, enabling the use of inexpensive and renewable feedstocks.
{"title":"Novel routes for bioproduction of delta lactone aroma compounds","authors":"Sonali Srivastava , Aakash Chandramouli , Payal Gupta , Abdur Rahman Manzer , Rahul Choudhury , D. Srinivasa Reddy , Syed Shams Yazdani , Siddhesh S. Kamat , Debasisa Mohanty , Vinay K. Nandicoori , Rajesh S. Gokhale","doi":"10.1016/j.ymben.2026.01.005","DOIUrl":"10.1016/j.ymben.2026.01.005","url":null,"abstract":"<div><div>Delta lactones are fatty acid-derived aroma compounds that hold tremendous commercial value. As consumer demand for natural flavours continues to rise, the bioproduction of δ-lactones, including δ-decalactone and δ-dodecalactone, is attracting substantial interest. Our study brings forth a novel approach to the bioproduction of δ-lactones from glucose, deviating from existing methods that primarily rely on the biotransformation of fatty acids. The high cost of fatty acid raw material constrains the commercial viability of this traditional approach. We engineered surface-lipid producing type I polyketide synthase (PKS) from <em>Mycobacterium smegmatis</em> by incorporating macrolactone thioesterase (TE) domain. Two out of three fusion constructs produced an appropriately engineered PKS-TE fusion protein that facilitated the synthesis of δ-lactones. When grown on glucose as the sole carbon source, recombinant <em>E. coli</em> expressing the engineered PKS-TE fusion protein successfully made δ-lactones ranging from C8-C18 acyl chains. Our research further highlights the potential of <em>Mycobacterium smegmatis</em> as a cell factory for producing fatty acid-based δ-lactones. By genetically designing and engineering <em>Mycobacterium smegmatis</em> to express PKS-TE fusion protein, we achieved bioproduction of various δ-lactones. Batch fermentation of the engineered <em>E. coli</em> strain fed with 2 % glucose produced 786 mg/L of δ-dodecalactone and 444 mg/L of δ-decalactone at 120 h, underscoring the efficacy of our approach. Thus, this study is the first to demonstrate a methodology for redirecting primary metabolic intermediates towards δ-lactone biosynthesis in engineered bacteria, enabling the use of inexpensive and renewable feedstocks.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 295-304"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145962523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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