Pub Date : 2021-12-01DOI: 10.1016/j.mec.2021.e00176
Kristina Stephens , Fauziah Rahma Zakaria , Eric VanArsdale , Gregory F. Payne , William E. Bentley
There is much to be gained by enabling electronic interrogation and control of biological function. While the benefits of bioelectronics that rely on potential-driven ionic flows are well known (electrocardiograms, defibrillators, neural prostheses, etc) there are relatively few advances targeting nonionic molecular networks, including genetic circuits. Redox activities combine connectivity to electronics with the potential for specific genetic control in cells. Here, electrode-generated hydrogen peroxide is used to actuate an electrogenetic “relay” cell population, which interprets the redox cue and synthesizes a bacterial signaling molecule (quorum sensing autoinducer AI-1) that, in turn, signals increased growth rate in a second population. The dramatically increased growth rate of the second population is enabled by expression of a phosphotransferase system protein, HPr, which is important for glucose transport. The potential to electronically modulate cell growth via direct genetic control will enable new opportunities in the treatment of disease and manufacture of biological therapeutics and other molecules.
{"title":"Electronic signals are electrogenetically relayed to control cell growth and co-culture composition","authors":"Kristina Stephens , Fauziah Rahma Zakaria , Eric VanArsdale , Gregory F. Payne , William E. Bentley","doi":"10.1016/j.mec.2021.e00176","DOIUrl":"10.1016/j.mec.2021.e00176","url":null,"abstract":"<div><p>There is much to be gained by enabling electronic interrogation and control of biological function. While the benefits of bioelectronics that rely on potential-driven ionic flows are well known (electrocardiograms, defibrillators, neural prostheses, etc) there are relatively few advances targeting nonionic molecular networks, including genetic circuits. Redox activities combine connectivity to electronics with the potential for specific genetic control in cells. Here, electrode-generated hydrogen peroxide is used to actuate an electrogenetic “relay” cell population, which interprets the redox cue and synthesizes a bacterial signaling molecule (quorum sensing autoinducer AI-1) that, in turn, signals increased growth rate in a second population. The dramatically increased growth rate of the second population is enabled by expression of a phosphotransferase system protein, HPr, which is important for glucose transport. The potential to electronically modulate cell growth via direct genetic control will enable new opportunities in the treatment of disease and manufacture of biological therapeutics and other molecules.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00176"},"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.e00176","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39058216","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}
Pub Date : 2021-12-01DOI: 10.1016/j.mec.2021.e00182
Bingyu Kuang , Venkata Gayatri Dhara , Duc Hoang , Jack Jenkins , Pranay Ladiwala , Yanglan Tan , Scott A. Shaffer , Shaun C. Galbraith , Michael J. Betenbaugh , Seongkyu Yoon
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.
哺乳动物细胞在生长和生产过程中消耗大量的营养物质。然而,内源性代谢效率低下往往阻止细胞充分利用营养来支持生长和蛋白质生产。相反,大量的营养物质被转移到细胞外积累的废物副产品和代谢物中,进一步抑制了增殖和蛋白质合成。本研究采用基于LC-MS/MS的代谢组学方法筛选中国仓鼠卵巢(CHO)细胞外代谢物。由细胞代谢效率低下引起的8种已确定的抑制性代谢物中有6种以前未在CHO细胞中研究过:乌头酸、2-羟基异己酸、甲基琥珀酸、单磷酸胞苷、葫芦巴碱和n-乙酰腐胺。当补充回补料批培养时,观察到每种代谢物存在时细胞生长显著减少,所有鉴定的代谢物都显示影响模型分泌抗体的糖基化,其中7种也降低CHO细胞生产力(滴度),所有8种都抑制单半乳糖化双天线(G1F)和双天线半乳糖化(G2F) n -聚糖的形成。这些抑制性代谢物进一步影响细胞的代谢,导致CHO细胞生长和比产率显著降低(最大降幅分别为27.2%和40.6%)。深入的途径分析表明,当细胞利用葡萄糖等主要能量来源和选择氨基酸(色氨酸、精氨酸、异亮氨酸和亮氨酸)进行生长、维持和蛋白质生产时,这些代谢物就会产生。此外,这些新的抑制性代谢物被观察到在多种CHO细胞系(CHO - k1和CHO- gs)以及HEK293细胞系中积累。本研究提供了一种强大而全面的方法,将全球代谢组学分析纳入细胞培养研究,以阐明和结构验证参与生物制药生产中生长,生产和翻译后修饰关键代谢途径的新型代谢物。
{"title":"Identification of novel inhibitory metabolites and impact verification on growth and protein synthesis in mammalian cells","authors":"Bingyu Kuang , Venkata Gayatri Dhara , Duc Hoang , Jack Jenkins , Pranay Ladiwala , Yanglan Tan , Scott A. Shaffer , Shaun C. Galbraith , Michael J. Betenbaugh , 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}
Pub Date : 2021-12-01DOI: 10.1016/j.mec.2021.e00175
Anton Puzorjov , Katherine E. Dunn , Alistair J. McCormick
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 Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an ‘Olive’ mutant strain of Synechocystis 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 T. elongatus, 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 T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.
{"title":"Production of thermostable phycocyanin in a mesophilic cyanobacterium","authors":"Anton Puzorjov , Katherine E. Dunn , 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<sup>−1</sup> 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}
Pub Date : 2021-12-01DOI: 10.1016/j.mec.2021.e00185
Jianhua Li , Fanglin Xu , Dongni Ji , Chenfei Tian , Yuwei Sun , Ishmael Mutanda , Yuhong Ren , Yong Wang
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 Glycine max) and GuCHR (chalcone reductase from Glycyrrhizza uralensis) 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 Medicago sativa), 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 Escherichia coli. 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.
{"title":"Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in Escherichia coli","authors":"Jianhua Li , Fanglin Xu , Dongni Ji , Chenfei Tian , Yuwei Sun , Ishmael Mutanda , Yuhong Ren , 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}
Pub Date : 2021-06-01DOI: 10.1016/j.mec.2021.e00162
David C. Garcia , Jaime Lorenzo N. Dinglasan , Him Shrestha , Paul E. Abraham , Robert L. Hettich , Mitchel J. Doktycz
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.
{"title":"A lysate proteome engineering strategy for enhancing cell-free metabolite production","authors":"David C. Garcia , Jaime Lorenzo N. Dinglasan , Him Shrestha , Paul E. Abraham , Robert L. Hettich , 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}
Pub Date : 2021-06-01DOI: 10.1016/j.mec.2021.e00168
Ruihua Zhang, Yan Zhang, Jian Wang, Yaping Yang, Yajun Yan
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.
{"title":"Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation","authors":"Ruihua Zhang, Yan Zhang, Jian Wang, Yaping Yang, 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}
Pub Date : 2021-06-01DOI: 10.1016/j.mec.2021.e00171
Dauenpen Meesapyodsuk , Yi Chen , Shengjian Ye , Robert G. Chapman , Xiao Qiu
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 Eranthis hyemalis 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 Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 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 sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in the effective incorporation of DDA and DTA at the sn-2 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.
{"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 , Yi Chen , Shengjian Ye , Robert G. Chapman , 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}
Pub Date : 2021-06-01DOI: 10.1016/j.mec.2021.e00163
Henna Mustila , Amit Kugler, Karin Stensjö
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 Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD 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 Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD 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 RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.
蓝藻可以作为直接光养转换二氧化碳的平台,以生产几种碳中性生物燃料。异丁烯是一种在蓝藻中产生光生物学的有前途的化合物。作为一种挥发性化合物,异丁烯会迅速逃离细胞,而不会在生长培养基中形成毒性水平,也不会被细胞膜捕获。与液体生物燃料不同,气态异丁烯可以从顶空收集,从而避免了从培养基或细胞中提取化学物质的昂贵费用。在这里,我们研究了一种适用于光自养寄主的异丁烯合成途径。首先,我们在大肠杆菌中表达褐家鼠α-酮异己酸双加氧酶(RnKICD)。我们发现纯化的RnKICD以α-酮异己酸酯为底物形成异丁烯的速率为104.6±9 ng (mg蛋白)-1 min-1。通过引入RnKICD酶,我们进一步证明了蓝细菌synnechocystis sp. PCC 6803中异丁烯的产生。异源表达RnKICD的胞囊菌产生91 ng l−1 OD750−1 h−1。因此,我们展示了一个新的可持续平台的蓝藻生产的一个重要的构建块化学品,异丁烯。这些结果表明,RnKICD可以通过蛋白质和代谢工程的努力进一步优化合成异丁烯途径。
{"title":"Isobutene production in Synechocystis sp. PCC 6803 by introducing α-ketoisocaproate dioxygenase from Rattus norvegicus","authors":"Henna Mustila , Amit Kugler, 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 CO<sub>2</sub> 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)<sup>-1</sup> min<sup>-1</sup> 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<sup>−1</sup> OD<sub>750</sub><sup>−1</sup> h<sup>−1</sup>. 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}