Metabolic Engineering Communications最新文献

{"title":"Erratum regarding previously published articles in volumes 9, 10 and 11","authors":"","doi":"10.1016/j.mec.2021.e00186","DOIUrl":"10.1016/j.mec.2021.e00186","url":null,"abstract":"","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00186"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569586/pdf/main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39613167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of novel inhibitory metabolites and impact verification on growth and protein synthesis in mammalian cells","authors":"Bingyu Kuang ,&nbsp;Venkata Gayatri Dhara ,&nbsp;Duc Hoang ,&nbsp;Jack Jenkins ,&nbsp;Pranay Ladiwala ,&nbsp;Yanglan Tan ,&nbsp;Scott A. Shaffer ,&nbsp;Shaun C. Galbraith ,&nbsp;Michael J. Betenbaugh ,&nbsp;Seongkyu Yoon","doi":"10.1016/j.mec.2021.e00182","DOIUrl":"10.1016/j.mec.2021.e00182","url":null,"abstract":"<div><p>Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO–K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00182"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00182","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39416572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of thermostable phycocyanin in a mesophilic cyanobacterium","authors":"Anton Puzorjov ,&nbsp;Katherine E. Dunn ,&nbsp;Alistair J. McCormick","doi":"10.1016/j.mec.2021.e00175","DOIUrl":"10.1016/j.mec.2021.e00175","url":null,"abstract":"<div><p>Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium <em>Thermosynechococcus elongatus</em> BP-1 (<em>cpcBACD</em>) is functional in the mesophile <em>Synechocystis</em> sp. PCC 6803. Expression of <em>cpcBACD</em> in an ‘Olive’ mutant strain of <em>Synechocystis</em> lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g⁻¹ DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from <em>T. elongatus</em>, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from <em>T. elongatus</em> in <em>Synechocystis</em> cultures. Our model showed that the higher yields and lower cultivation temperatures of <em>Synechocystis</em> resulted in a 3.5-fold increase in energy efficiency compared to <em>T. elongatus</em>, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00175"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00175","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39106410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum regarding missing Declaration of competing interest statements in previously published articles","authors":"","doi":"10.1016/j.mec.2021.e00189","DOIUrl":"https://doi.org/10.1016/j.mec.2021.e00189","url":null,"abstract":"","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00189"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030121000298/pdfft?md5=40f55f2d13a05cd5e89ecc924b894482&pid=1-s2.0-S2214030121000298-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136696720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in Escherichia coli","authors":"Jianhua Li ,&nbsp;Fanglin Xu ,&nbsp;Dongni Ji ,&nbsp;Chenfei Tian ,&nbsp;Yuwei Sun ,&nbsp;Ishmael Mutanda ,&nbsp;Yuhong Ren ,&nbsp;Yong Wang","doi":"10.1016/j.mec.2021.e00185","DOIUrl":"10.1016/j.mec.2021.e00185","url":null,"abstract":"<div><p>5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues. In this study, GmCHS7 (chalcone synthase from <em>Glycine max</em>) and GuCHR (chalcone reductase from <em>Glycyrrhizza uralensis</em>) were preliminarily utilized to improve the CHR ratio (CHR product to total CHS product). The use of this enzyme combination improved the final CHR ratio from 39.7% to 50.3%. For further optimization, a protein-protein interaction strategy was employed, basing on the spatial adhesion of GmCHS7:PDZ and GuCHR:PDZlig. This strategy further increased the ratio towards the CHR-derived product (54.7%), suggesting partial success of redirecting metabolic flux towards the reduced branch. To further increase the total carbon metabolic flux, 15 protein scaffolds were programmed with stoichiometric arrangement of the three sequential catalysts GmCHS7, GuCHR and MsCHI (chalcone isomerase from <em>Medicago sativa</em>), resulting in a 1.4-fold increase in total flavanone production, from 69.4 mg/L to 97.0 mg/L in shake flasks. The protein self-assembly strategy also improved the production and direction of the lineage-specific compounds 7,4′-dihydroxyflavone and daidzein in <em>Escherichia coli</em>. This study presents a significant advancement of 5-deoxy(iso)flavonoid production and provides the foundation for production of value-added 5-deoxy(iso)flavonoids in microbial hosts.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00185"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f2/32/main.PMC8488244.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39503749","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A lysate proteome engineering strategy for enhancing cell-free metabolite production","authors":"David C. Garcia ,&nbsp;Jaime Lorenzo N. Dinglasan ,&nbsp;Him Shrestha ,&nbsp;Paul E. Abraham ,&nbsp;Robert L. Hettich ,&nbsp;Mitchel J. Doktycz","doi":"10.1016/j.mec.2021.e00162","DOIUrl":"10.1016/j.mec.2021.e00162","url":null,"abstract":"<div><p>Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"12 ","pages":"Article e00162"},"PeriodicalIF":5.2,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00162","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25344116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation","authors":"Ruihua Zhang,&nbsp;Yan Zhang,&nbsp;Jian Wang,&nbsp;Yaping Yang,&nbsp;Yajun Yan","doi":"10.1016/j.mec.2021.e00168","DOIUrl":"10.1016/j.mec.2021.e00168","url":null,"abstract":"<div><p>Trans-regulating elements such as noncoding RNAs are crucial in modifying cells, and has shown broad application in synthetic biology, metabolic engineering and RNA therapies. Although effective, titration of the regulatory levels of such elements is less explored. Encouraged by the need of fine-tuning cellular functions, we studied key parameters of the antisense RNA design including oligonucleotide length, targeting region and relative dosage to achieve differentiated inhibition. We determined a 30-nucleotide configuration that renders efficient and robust inhibition. We found that by targeting the core RBS region proportionally, quantifiable inhibition levels can be rationally obtained. A mathematic model was established accordingly with refined energy terms and successfully validated by depicting the inhibition levels for genomic targets. Additionally, we applied this fine-tuning approach for 4-hydroxycoumarin biosynthesis by simultaneous and quantifiable knockdown of multiple targets, resulting in a 3.58-fold increase in titer of the engineered strain comparing to that of the non-regulated. We believe the developed tool is broadly compatible and provides an extra layer of control in modifying living systems.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"12 ","pages":"Article e00168"},"PeriodicalIF":5.2,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00168","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25477408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Co-expressing Eranthis hyemalis lysophosphatidic acid acyltransferase 2 and elongase improves two very long chain polyunsaturated fatty acid production in Brassica carinata","authors":"Dauenpen Meesapyodsuk ,&nbsp;Yi Chen ,&nbsp;Shengjian Ye ,&nbsp;Robert G. Chapman ,&nbsp;Xiao Qiu","doi":"10.1016/j.mec.2021.e00171","DOIUrl":"10.1016/j.mec.2021.e00171","url":null,"abstract":"<div><p>Docosadienoic acid (DDA, 22:2–13,16) and docosatrienoic acid (DTA, 22:3–13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant <em>Eranthis hyemalis</em> can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop <em>Brassica carinata</em> produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the <em>sn-2</em> position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the <em>sn</em>-2 position of triacylglycerols was identified from <em>E. hyemalis</em>. RT-PCR analysis showed that it was preferentially expressed in developing seeds where <em>EhELO1</em> was exclusively expressed in <em>E. hyemalis</em>. Seed specific expression of <em>EhLPAAT2</em> along with <em>EhELO1</em> in <em>B. carinata</em> resulted in the effective incorporation of DDA and DTA at the <em>sn-2</em> position of TAGs, thereby increasing the total amount of DDA and DTA in transgenic seeds. To our knowledge, this is the first plant LPAAT that can incorporate VLCPUFAs into TAGs. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"12 ","pages":"Article e00171"},"PeriodicalIF":5.2,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00171","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38941339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isobutene production in Synechocystis sp. PCC 6803 by introducing α-ketoisocaproate dioxygenase from Rattus norvegicus","authors":"Henna Mustila ,&nbsp;Amit Kugler,&nbsp;Karin Stensjö","doi":"10.1016/j.mec.2021.e00163","DOIUrl":"10.1016/j.mec.2021.e00163","url":null,"abstract":"<div><p>Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO₂ to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from <em>Rattus norvegicus</em> (<em>Rn</em>KICD) in <em>Escherichia coli</em>. We discovered isobutene formation with the purified <em>Rn</em>KICD with the rate of 104.6 ​± ​9 ​ng (mg protein)^-1 min^-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium <em>Synechocystis</em> sp. PCC 6803 by introducing the <em>Rn</em>KICD enzyme. <em>Synechocystis</em> strain heterologously expressing the <em>Rn</em>KICD produced 91 ​ng ​l⁻¹ OD₇₅₀⁻¹ ​h⁻¹. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that <em>Rn</em>KICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"12 ","pages":"Article e00163"},"PeriodicalIF":5.2,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00163","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25344117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of metabolic network disruption in engineered microbial hosts due to enzyme promiscuity","authors":"Vladimir Porokhin ,&nbsp;Sara A. Amin ,&nbsp;Trevor B. Nicks ,&nbsp;Venkatesh Endalur Gopinarayanan ,&nbsp;Nikhil U. Nair ,&nbsp;Soha Hassoun","doi":"10.1016/j.mec.2021.e00170","DOIUrl":"10.1016/j.mec.2021.e00170","url":null,"abstract":"<div><p>Increasing understanding of metabolic and regulatory networks underlying microbial physiology has enabled creation of progressively more complex synthetic biological systems for biochemical, biomedical, agricultural, and environmental applications. However, despite best efforts, confounding phenotypes still emerge from unforeseen interplay between biological parts, and the design of robust and modular biological systems remains elusive. Such interactions are difficult to predict when designing synthetic systems and may manifest during experimental testing as inefficiencies that need to be overcome. Transforming organisms such as <em>Escherichia coli</em> into microbial factories is achieved via several engineering strategies, used individually or in combination, with the goal of maximizing the production of chosen target compounds. One technique relies on suppressing or overexpressing selected genes; another involves introducing heterologous enzymes into a microbial host. These modifications steer mass flux towards the set of desired metabolites but may create unexpected interactions. In this work, we develop a computational method, termed <u>M</u>etabolic <u>D</u>isruption Work<u>flow</u> (<em>MDFlow</em>), for discovering interactions and network disruptions arising from enzyme promiscuity – the ability of enzymes to act on a wide range of molecules that are structurally similar to their native substrates. We apply <em>MDFlow</em> to two experimentally verified cases where strains with essential genes knocked out are rescued by interactions resulting from overexpression of one or more other genes. We demonstrate how enzyme promiscuity may aid cells in adapting to disruptions of essential metabolic functions. We then apply <em>MDFlow</em> to predict and evaluate a number of putative promiscuous reactions that can interfere with two heterologous pathways designed for 3-hydroxypropionic acid (3-HP) production. Using <em>MDFlow</em>, we can identify putative enzyme promiscuity and the subsequent formation of unintended and undesirable byproducts that are not only disruptive to the host metabolism but also to the intended end-objective of high biosynthetic productivity and yield. As we demonstrate, <em>MDFlow</em> provides an innovative workflow to systematically identify incompatibilities between the native metabolism of the host and its engineered modifications due to enzyme promiscuity.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"12 ","pages":"Article e00170"},"PeriodicalIF":5.2,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00170","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25586461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}