Journal of biotechnology最新文献

{"title":"Discovery and characterization of NADH oxidases for selective sustainable synthesis of 5-hydroxymethylfuran carboxylic acid.","authors":"Karishma Shah, Daniel Kracher, Peter Macheroux, Silvia Wallner, André Pick, Robert Kourist","doi":"10.1016/j.jbiotec.2024.11.009","DOIUrl":"10.1016/j.jbiotec.2024.11.009","url":null,"abstract":"<p><p>Efficient regeneration of NAD⁺ remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that AdNox, GdNox, CmNox, and LvNox exhibit V_max values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97 % in the presence of 0.1 mM NAD and 10 mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis.</p>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":" ","pages":"18-28"},"PeriodicalIF":4.1,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739743","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}

{"title":"Mechanism of rhamnolipid promoting the degradation of polycyclic aromatic hydrocarbons by gram-positive bacteria—Enhance transmembrane transport and electron transfer","authors":"Bo Zhang ,&nbsp;Lei Wang ,&nbsp;Zhenjun Diwu ,&nbsp;Maiqian Nie ,&nbsp;Hongyun Nie","doi":"10.1016/j.jbiotec.2024.11.010","DOIUrl":"10.1016/j.jbiotec.2024.11.010","url":null,"abstract":"<div><div>In this study, the Gram-positive bacterium <em>Bacillus licheniformis</em> T5 was utilized to investigate the impact of rhamnolipid on cell membrane and cell wall, as well as enzyme activity and electron transfer rate within cells. Results indicated that at the optimal concentration of rhamnolipid (200 mg/L), the cell membrane protein and cell wall peptidoglycan content of T5 decreased significantly. Infrared spectrum analysis and ultrastructure observations confirmed these findings, revealing noticeable changes in cell morphology in the presence of rhamnolipid. Specifically, cell folds increased, cell wall texture loosened, thickness decreased sharply, transmembrane channels appeared, and the plasma wall slightly separated. These alterations likely contributed to the increased permeability of the cell membrane. Furthermore, rhamnolipid accelerated the electron transfer rate in T5 cells, enhancing oxidoreductase activity. This study elucidates the mechanism through which rhamnolipid promotes the degradation of polycyclic aromatic hydrocarbons by Gram-positive bacteria, focusing on transmembrane transport and catalytic metabolism.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 51-60"},"PeriodicalIF":4.1,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142706753","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}

{"title":"Engineering Saccharomyces boulardii for cell surface display of heterologous protein","authors":"Jamin Shin ,&nbsp;Gayoung Lee ,&nbsp;Won-Jae Chi ,&nbsp;Sujeong Park ,&nbsp;Yong-Su Jin ,&nbsp;Soo Rin Kim","doi":"10.1016/j.jbiotec.2024.11.013","DOIUrl":"10.1016/j.jbiotec.2024.11.013","url":null,"abstract":"<div><div><em>Saccharomyces boulardii</em> and <em>Saccharomyces cerevisiae</em> share over 99 % genetic similarity yet exhibit distinct metabolic traits. While the cell surface display system of <em>S. cerevisiae</em> is well-documented, the equivalent system in <em>S. boulardii</em> has yet to be fully characterized. This study investigates the cell surface display system of <em>S. boulardii</em> for the expression of a heterologous protein using different anchor proteins. Six strains expressing the enhanced green fluorescent protein (Egfp) and an anchor protein as a fusion protein were constructed to visualize the cell surface display system. Then a heterologous endo-inulinase protein was expressed with selected anchor proteins through fluorescence intensity comparison. Analysis by fluorescence microscopy revealed that the anchor protein Sed1 exhibited the highest fluorescence intensity. Furthermore, expressed selected anchor proteins and heterologous protein, endo-inulinase, the engineered strain could degrade and consume almost inulin in 72 h. Through endo-inulinase expression, we confirmed that not only Egfp but also heterologous protein is well expressed, and we successfully built an <em>S. boulardii</em> cell surface display system.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 44-50"},"PeriodicalIF":4.1,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692964","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}

{"title":"Comparative analysis of substrate- and regio-selectivity of HpaB monooxygenases and their application to hydroxydaidzein synthesis","authors":"Sachiko Watanabe ,&nbsp;Hideki Kato ,&nbsp;Kento Yoshinaga ,&nbsp;Akiko Kohara ,&nbsp;Yuichi Ukawa ,&nbsp;Akinobu Matsuyama ,&nbsp;Toshiki Furuya","doi":"10.1016/j.jbiotec.2024.11.012","DOIUrl":"10.1016/j.jbiotec.2024.11.012","url":null,"abstract":"<div><div>4-Hydroxyphenylacetate 3-hydroxylase (HpaB) has high potential for use in polyphenol synthesis via <em>ortho</em>-hydroxylation. Although the HpaB enzymes from <em>Pseudomonas aeruginosa</em> (PaHpaB) and <em>Escherichia coli</em> (EcHpaB) have been well studied, few studies have compared their activity and substrate selectivity. Thus, which HpaB is optimal for use in the biotechnological production of polyphenols is unclear. In this study, we performed a comparative analysis of the substrate- and regio-selectivity of PaHpaB, EcHpaB, and the recently discovered enzyme from <em>Rhodococcus opacus</em> (RoHpaB). The activity of these enzymes was first compared toward representative aromatic substrates. PaHpaB and EcHpaB exhibited very similar catalytic activity toward <em>p</em>-coumaric acid and tyrosol with one benzene ring, whereas PaHpaB exhibited greater activity than EcHpaB toward resveratrol and naringenin with two benzene rings. These results suggest that PaHpaB is superior to EcHpaB in converting bulky compounds. Furthermore, PaHpaB also exhibited catalytic activity toward a flavonoid, daidzein (7,4′-dihydroxyisoflavone), whereas EcHpaB did not. RoHpaB also exhibited strong activity toward daidzein in addition to other aromatic substrates. Interestingly, PaHpaB hydroxylated the 6-position of daidzein, whereas RoHpaB hydroxylated the 3′-position. PaHpaB and RoHpaB enabled the facile synthesis of not only 6-hydroxydaidzein and 3′-hydroxydaidzein but also 6,3′-dihydroxydaidzein via the cascade reaction. This study is the first to demonstrate synthesis of hydroxydaidzeins using HpaB enzymes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 61-66"},"PeriodicalIF":4.1,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692963","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}

{"title":"Digital holographic microscopy is suitable for lipid accumulation analysis in single cells of Yarrowia lipolytica","authors":"Simon Carl-Philipp Briel ,&nbsp;Nicolas Feuser ,&nbsp;Eva Johanna Moldenhauer ,&nbsp;Johannes Kabisch ,&nbsp;Peter Neubauer ,&nbsp;Stefan Junne","doi":"10.1016/j.jbiotec.2024.11.011","DOIUrl":"10.1016/j.jbiotec.2024.11.011","url":null,"abstract":"<div><div>Digital holographic microscopy (DHM) is a label-free analytical technique for the determination of the cells’ volume and their cytosolic refractive index. Here, we demonstrate the suitability of DHM for the quantification of total lipid accumulation in the oleaginous yeast <em>Yarrowia lipolytica</em>. Presently, microbial lipids are gaining increasing attention due to their nutritional value in feed and food applications. Their microbiological synthesis in algae and yeast is subject to optimization studies, which necessitates rapid quantification of total lipids for faster progress and the possibility of process control. So far, quantification of the total intracellular long-chain fatty acid concentration in yeast cells is time-consuming though when common chromatography for a volumetric analysis or staining and flow cytometry for a single-cell based analysis are used. This study, however, demonstrates that 3D-DHM facilitates a quasi-real-time measurement that allows for a rapid quantification of total intracellular lipid accumulation on a single-cell level without cell staining. Data from wild-type and lipid overproducing <em>Y. lipolytica</em> strains with specific yields of long-chain fatty acids in a range between 70 and 360 mg/gCDW show a good correlation with the optical volume determined by DHM, as the total lipid accumulation in the cell is typically well correlated with the long-chain fatty acid concentration. The results further correlate with data obtained from gas chromatography and flow cytometry of Nile Red-stained cells, which proves the reliability of DHM for lipid quantification in <em>Y. lipolytica</em>.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 32-43"},"PeriodicalIF":4.1,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648166","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}

{"title":"A pump-free microfluidic co-culture system for investigating NK cell-tumor spheroid interactions in flow conditions","authors":"Yuanyuan Xie ,&nbsp;Ke Ning ,&nbsp;Wen Sun ,&nbsp;Lingke Feng ,&nbsp;Yirong Chen ,&nbsp;Wei Sun ,&nbsp;Yan Li ,&nbsp;Ling Yu","doi":"10.1016/j.jbiotec.2024.11.008","DOIUrl":"10.1016/j.jbiotec.2024.11.008","url":null,"abstract":"<div><div>Natural killer (NK) cells are pivotal in immunotherapy due to their potent tumor-targeting capabilities. However, accessible <em>in vitro</em> 3D dynamic models for evaluating Tumor Infiltrating Natural Killer Cells (TINKs) remain scarce. This study addresses this gap by developing a novel pump-free microfluidic chip to investigate the interactions between NK-92 cells and prostate DU 145 tumor spheroids. The platform facilitates the separation of free NKs and TINKs for subtype characterization. The design integrates multiple planes with a multi-layer paper scaffold to accommodate tumor spheroids, allowing NK-92 cells to traverse Matrigel-coated barriers that mimic the extracellular matrix. The dual-channel pump-free device enables unidirectional circulation of NK-92 cells, allowing analysis of tumor spheroid movement and NK-92 cell interactions under flow conditions. Results demonstrate continuous fluid circulation in the dual-channel device by rocking the platform at tilt angles of 21° and 15°. Tumor spheroids show- enhanced migration under flow conditions compared to static culture. Although spheroids recruit slightly more NK-92 cells under flow conditions, CD56 and CD16 receptor expression on IL-2-activated free NK-92 cells and tumor-infiltrating NK-92 cells matches <em>in vivo</em> patterns in dynamic cultures. These findings suggest that tumor cells and fluid dynamics significantly influence NK cell subtypes. This pump-free microfluidic platform is a functional tool for simulating and studying immune cell-tumor interactions, providing valuable insights into NK cell dynamics with tumor spheroids in physiologically relevant environments.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 11-21"},"PeriodicalIF":4.1,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142644206","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}

{"title":"Engineering Saccharomyces cerevisiae for continuous secretory production of hEGF in biofilm","authors":"Kaiqi Zhi ,&nbsp;Xiang Zhou ,&nbsp;Tianping Gao ,&nbsp;Kehan Liu ,&nbsp;Zhenyu Wang ,&nbsp;Yafan Cai ,&nbsp;Zhi Wang ,&nbsp;Shilei Wang ,&nbsp;Jinle Liu ,&nbsp;Dong Liu ,&nbsp;Hanjie Ying","doi":"10.1016/j.jbiotec.2024.11.007","DOIUrl":"10.1016/j.jbiotec.2024.11.007","url":null,"abstract":"<div><div>Human epidermal growth factor (hEGF) plays a crucial role in promoting cell growth and has various clinical applications. Due to limited natural sources and the high cost of chemical synthesis, researchers are now exploring genetic engineering as a potential method for hEGF production. In this particular study, a novel hEGF expression system was developed using <em>Saccharomyces cerevisiae</em>. This system involved optimizing the promoter and signal peptide and deleting protease-coding genes <em>PEP4</em>, <em>PRB1</em>, and <em>YAP3</em>, overexpressing chaperones <em>KAR2</em> and <em>PDI1</em> in the protein secretion pathway, which led to a 2.01-fold increase in hEGF production compared to the wild type strain. Furthermore, biofilm-forming genes <em>FLO11</em> and <em>ALS3</em> were integrated to create a biofilm strain with adhesive properties. A biofilm-based immobilized continuous fermentation model was established to leverage the characteristics of this biofilm strain. Each batch of this model yielded 130 mg/L of hEGF, with a production efficiency of 2.71 mg/L/h - surpassing the production efficiency of traditional free fermentation (1.62 mg/L/h). This study presents a promising fermentation model for efficient hEGF production based on biofilm characteristics, offering valuable insights for the application of biofilm fermentation in the production of small molecule peptides.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"397 ","pages":"Pages 1-10"},"PeriodicalIF":4.1,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142644207","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}

{"title":"Hydrogen production capabilities of lichens micro-ecosystem under extreme salinity, crystalline salt exposure, and simulated Mars-like conditions","authors":"Maria Fanara ,&nbsp;Aikaterini Papazi ,&nbsp;Stergios Pirintsos ,&nbsp;Kiriakos Kotzabasis","doi":"10.1016/j.jbiotec.2024.11.004","DOIUrl":"10.1016/j.jbiotec.2024.11.004","url":null,"abstract":"<div><div>This work aims to demonstrate the extremophilic behavior of the lichen <em>Pleurosticta acetabulum</em> at extreme salinities, while maintaining its metabolic capacity to produce hydrogen. Lichen is a special micro-ecosystem that includes mostly a fungus and a green alga or cyanobacterium, as well as a microbiome. The peculiarity of this symbiotic system is its ability to dry out completely and stay inactive to survive harsh conditions. Lichens that had been dehydrated for six months revived quickly when rehydrated, restoring their photosynthetic efficiency and ability to produce hydrogen. The lichen microbiome was crucial for hydrogen production, especially through dark fermentation. The experiments of this work showed that lichen during its exposure to different salinity conditions (0 %NaCl – control, 3,5 %NaCl – sea salt concentration, 36 %NaCl – saturated salt concentration), but also after exposure to crystalline salt (100 %NaCl) could maintain the structure and the functionality of its photosynthetic apparatus. This was tested using chlorophyll a fluorescence induction measurements. Based on the results from gas chromatography with thermal conductivity detection (GC-TCD) used to determine hydrogen production, it was shown that despite being exposed to extreme salinity conditions, lichens maintained their ability to produce hydrogen. The experimental combination of lichen exposure to extreme salinities (up to 100 % NaCl), with an extreme atmosphere (100 % CO₂) and low atmospheric pressure (&lt;10mbar), simulating Mars conditions, highlighted the functional potential of the lichen for survival in a Mars-like environment. This lichen’s ability to withstand extreme conditions and to produce large amounts of hydrogen, makes it a promising candidate for future biotechnological applications, even in challenging environments like Mars, opening new astrobiological and astrobiotechnological perspectives.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"396 ","pages":"Pages 171-179"},"PeriodicalIF":4.1,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621003","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}

{"title":"Structure-guided engineering of 4-coumarate: CoA ligase for efficient production of rosmarinic acid in Saccharomyces cerevisiae","authors":"Xiuqi Zhou ,&nbsp;Jiayan Du ,&nbsp;Jinyuan Zhu ,&nbsp;Xueqing Pang ,&nbsp;Xinjian Yin ,&nbsp;Pingping Zhou","doi":"10.1016/j.jbiotec.2024.11.006","DOIUrl":"10.1016/j.jbiotec.2024.11.006","url":null,"abstract":"<div><div>The utilization of genetically modified microbial cells for rosmarinic acid (RA) production is gaining increased attention as a cost-effective and sustainable approach. However, the substrate promiscuity of 4-coumarate: CoA ligase and RA synthase has been considered as a critical factor for low RA yields. In this study, we rationally engineered the substrate preference of 4-coumarate: CoA ligase (OPc4CL2) from <em>Petroselinum crispum</em>, resulting in a significant enhancement in RA production. Particularly, the introduction of the Y240C mutation led to a remarkable 176 % increase in RA yield. Subsequent enzymatic analysis of OPc4CL2 variants revealed diminished activity towards <em>p</em>-coumaric acid, resulting in insufficient time for the transformation of <em>p</em>-coumaric acid to 4-coumaroyl CoA to generate byproduct. Furthermore, to minimize the formation of undesired byproducts, the overexpression of 4-hydroxyphenylacetate 3-monooxygenase (OHpaB) and NADPH-flavin oxidoreductase (HpaC) was carried out to facilitate the conversion of <em>p</em>-coumaric acid to caffeic acid and 4-hydroxyphenyllactate to salvianic acid A, thus achieving a significant increase in RA yield of up to 329.9 mg/L (16.5 mg/g yield on glucose) in shake-flask cultivation. Finally, the engineered strain YRA113–24BHM achieved a notable RA production of 3.6 g/L (about 20.2 mg/g yield on glucose) by fed-batch fermentation. This study serves as a foundation for the sustainable biosynthesis of RA and other caffeic acid derivatives.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"396 ","pages":"Pages 140-149"},"PeriodicalIF":4.1,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621085","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}

{"title":"Optimisation of coumaric acid production from aromatic amino acids in Kluyveromyces marxianus","authors":"Joel A. Akinola ,&nbsp;Arun S. Rajkumar ,&nbsp;John P. Morrissey","doi":"10.1016/j.jbiotec.2024.11.002","DOIUrl":"10.1016/j.jbiotec.2024.11.002","url":null,"abstract":"<div><div>Yeasts are attractive hosts for the production of heterologous products due to their genetic tractability and relative ease of growth. While the baker’s yeast <em>Saccharomyces cerevisiae</em> is a powerful workhorse of the biotechnology industry, the species has metabolic limitations and it is critical that we develop alternative platforms that will facilitate the development of bioprocesses that rely on sustainable feedstocks. In this study, we used synthetic biology tools to construct coumaric acid–producing strains of <em>Kluyveromyces marxianus,</em> a yeast whose physiological traits render it attractive for biotechnology applications. Coumaric acid is a building block in the synthesis of many different families of aromatics and is a key precursor for the synthesis of complect phenylpropanoid molecules, including many flavours and aromas. The starting point for this work was a <em>K. marxianus</em> chassis strain that has increased flux towards the synthesis of tyrosine and phenylalanine, the aromatic amino acids that can serve as starting points for coumaric acid synthesis. Following principles of synthetic biology, a modular approach was taken to identify the best solution to different metabolic possibilities and these were then combined in different ways. For the first step, it was established that the route from phenylalanine was superior to that from tyrosine and the combined overexpression of <em>PlPAL</em>, <em>AtC4H</em> and <em>AtCPR1</em> delivered the highest yield of coumaric acid. Next, it was established that while Pdc5 and Aro10 both had phenylpyruvate decarboxylase activity, inactivation of <em>ARO10</em> was sufficient to prevent flux loss in the pathway. Since phenylalanine is the starting point, efforts were made to improve efficiency of its production. It was found that glutamate was a preferred nitrogen source for coumaric acid production, and this knowledge was used to engineer a strain that overexpressed <em>S. cerevisiae GDH1</em> and delivered higher yields of coumaric acid. Ultimately, this strategy led to the development of strains that has yields of up to 48 mg coumaric acid /g glucose. Strains were evaluated in bioreactors to investigate the effects of different process parameters. These analyses indicated that engineered strains face some redox balance challenges and further work will be required overcome these to develop strains that can perform well under industrial conditions.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"396 ","pages":"Pages 158-170"},"PeriodicalIF":4.1,"publicationDate":"2024-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621017","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}