Pub Date : 2021-06-01DOI: 10.1016/j.mec.2021.e00166
Ye Sol Jeong , Hyung-Keun Ku , Young-Joo Jung , Jae Kwang Kim , Kyoung Bok Lee , Ju-Kon Kim , Sun-Hyung Lim , Dongho Lee , Sun-Hwa Ha
Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.
{"title":"2A-linked bi-, tri-, and quad-cistrons for the stepwise biosynthesis of β-carotene, zeaxanthin, and ketocarotenoids in rice endosperm","authors":"Ye Sol Jeong , Hyung-Keun Ku , Young-Joo Jung , Jae Kwang Kim , Kyoung Bok Lee , Ju-Kon Kim , Sun-Hyung Lim , Dongho Lee , Sun-Hwa Ha","doi":"10.1016/j.mec.2021.e00166","DOIUrl":"10.1016/j.mec.2021.e00166","url":null,"abstract":"<div><p>Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from <em>Thosea asigna</em> virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"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.e00166","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25431233","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00130
Alyssa M. Worland , Jeffrey J. Czajka , Yun Xing , Willie F. Harper Jr. , Aryiana Moore , Zhengyang Xiao , Zhenlin Han , Yechun Wang , Wei Wen Su , Yinjie J. Tang
This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a β-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best β-carotene titer (121 ± 13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.
{"title":"Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock","authors":"Alyssa M. Worland , Jeffrey J. Czajka , Yun Xing , Willie F. Harper Jr. , Aryiana Moore , Zhengyang Xiao , Zhenlin Han , Yechun Wang , Wei Wen Su , Yinjie J. Tang","doi":"10.1016/j.mec.2020.e00130","DOIUrl":"10.1016/j.mec.2020.e00130","url":null,"abstract":"<div><p>This study employs biomass growth analyses and <sup>13</sup>C-isotope tracing to investigate lipid feedstock utilization by <em>Yarrowia lipolytica</em>. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a β-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best β-carotene titer (121 ± 13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of <em>Y. lipolytica</em> for the valorization of waste-derived lipid feedstock.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00130","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38082753","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00137
Yonghao Cui , Jianzhong He , Kun-Lin Yang , Kang Zhou
An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. In return, acetone produced by C. beijerinckii G117 was reduced into isopropanol by B. subtilis BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered B. subtilis. In the symbiotic system consisting of two strains, 1.7 g/L of acetone, 4.8 g/L of butanol, and 0.9 g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 g/L of acetone, 4.9 g/L of butanol, and 0.2 g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 g/L when we increased the inoculum size of B. subtilis BsADH2 and optimized other process parameters. With the Bacillus-Clostridium co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of Clostridium. This strategy of employing a recombinant Bacillus to co-culture with Clostridium should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.
{"title":"Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of Clostridium beijerinckii G117 and recombinant Bacillus subtilis 1A1","authors":"Yonghao Cui , Jianzhong He , Kun-Lin Yang , Kang Zhou","doi":"10.1016/j.mec.2020.e00137","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00137","url":null,"abstract":"<div><p>An engineered <em>B. subtilis</em> 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with <em>C. beijerinckii</em> G117 under an aerobic condition. During the fermentation on glucose, <em>B. subtilis</em> BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for <em>C. beijerinckii</em> G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by <em>B. subtilis</em> BsADH2 was re-assimilated by <em>C. beijerinckii</em> G117. In return, acetone produced by <em>C. beijerinckii</em> G117 was reduced into isopropanol by <em>B. subtilis</em> BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered <em>B. subtilis</em>. In the symbiotic system consisting of two strains, 1.7 g/L of acetone, 4.8 g/L of butanol, and 0.9 g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 g/L of acetone, 4.9 g/L of butanol, and 0.2 g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 g/L when we increased the inoculum size of <em>B. subtilis</em> BsADH2 and optimized other process parameters. With the <em>Bacillus</em>-<em>Clostridium</em> co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of <em>Clostridium</em>. This strategy of employing a recombinant <em>Bacillus</em> to co-culture with <em>Clostridium</em> should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00137","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91986220","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00144
Bruno Motta Nascimento, Nikhil U. Nair
Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.
{"title":"Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli","authors":"Bruno Motta Nascimento, Nikhil U. Nair","doi":"10.1016/j.mec.2020.e00144","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00144","url":null,"abstract":"<div><p>Poly-γ-glutamic acid (PGA) produced by many <em>Bacillus</em> species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the <em>Escherichia coli</em> membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In <em>E. coli</em>, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00144","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92060874","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00133
Jhonatan A. Hernandez-Valdes , Myrthe aan de Stegge , Jos Hermans , Johan Teunis , Rinke J. van Tatenhove-Pel , Bas Teusink , Herwig Bachmann , Oscar P. Kuipers
Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5–10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.
{"title":"Enhancement of amino acid production and secretion by Lactococcus lactis using a droplet-based biosensing and selection system","authors":"Jhonatan A. Hernandez-Valdes , Myrthe aan de Stegge , Jos Hermans , Johan Teunis , Rinke J. van Tatenhove-Pel , Bas Teusink , Herwig Bachmann , Oscar P. Kuipers","doi":"10.1016/j.mec.2020.e00133","DOIUrl":"10.1016/j.mec.2020.e00133","url":null,"abstract":"<div><p>Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, <em>Lactococcus lactis</em>, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some <em>L. lactis</em> strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type <em>L. lactis</em> strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated <em>L. lactis</em> IPLA838 strains with 5–10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00133","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38054684","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00146
Qin He , Patrycja Szczepańska , Tigran Yuzbashev , Zbigniew Lazar , Rodrigo Ledesma-Amaro
Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.
{"title":"De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia lipolytica","authors":"Qin He , Patrycja Szczepańska , Tigran Yuzbashev , Zbigniew Lazar , Rodrigo Ledesma-Amaro","doi":"10.1016/j.mec.2020.e00146","DOIUrl":"10.1016/j.mec.2020.e00146","url":null,"abstract":"<div><p>Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct <em>Yarrowia lipolytica</em> strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 g/L was reached. This study suggests that <em>Y. lipolytica</em> is a promising host for resveratrol production from glycerol.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00146","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38452381","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00141
Yanfeng Liu , Anqi Su , Rongzhen Tian , Jianghua Li , Long Liu , Guocheng Du
Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD, hag, and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD, hag, and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h-1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.
{"title":"Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production","authors":"Yanfeng Liu , Anqi Su , Rongzhen Tian , Jianghua Li , Long Liu , Guocheng Du","doi":"10.1016/j.mec.2020.e00141","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00141","url":null,"abstract":"<div><p><em>Bacillus subtilis</em> is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered <em>B. subtilis</em> with faster growth phenotype is lacking. Here, fast-growing <em>B. subtilis</em> were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain <em>B. subtilis</em> 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of <em>oppD</em>, <em>hag</em>, and <em>flgD</em> genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting <em>oppD</em>, <em>hag</em>, and <em>flgD</em> genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h<sup>-1</sup> in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing <em>B. subtilis</em> strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00141","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92060875","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00135
Chen Deng , Xueqin Lv , Jianghua Li , Yanfeng Liu , Guocheng Du , Long Liu
As a traditional amino acid producing bacterium, Corynebacterium glutamicum is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of C. glutamicum have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of zwf gene in C. glutamicum S9114 genome to improve the production of a typical nutraceutical N-acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in C. glutamicum.
{"title":"Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering","authors":"Chen Deng , Xueqin Lv , Jianghua Li , Yanfeng Liu , Guocheng Du , Long Liu","doi":"10.1016/j.mec.2020.e00135","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00135","url":null,"abstract":"<div><p>As a traditional amino acid producing bacterium, <em>Corynebacterium glutamicum</em> is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of <em>C. glutamicum</em> have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of <em>zwf</em> gene in <em>C. glutamicum</em> S9114 genome to improve the production of a typical nutraceutical <em>N</em>-acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in <em>C. glutamicum</em>.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00135","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92139338","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00151
Xiaoguang Fan , Tong Zhang , Yuanqing Ji , Jie Li , Keyi Long , Yue Yuan , Yanjun Li , Qingyang Xu , Ning Chen , Xixian Xie
L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-PT7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 g/g glucose and 2.72 g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.
{"title":"Pathway engineering of Escherichia coli for one-step fermentative production of L-theanine from sugars and ethylamine","authors":"Xiaoguang Fan , Tong Zhang , Yuanqing Ji , Jie Li , Keyi Long , Yue Yuan , Yanjun Li , Qingyang Xu , Ning Chen , Xixian Xie","doi":"10.1016/j.mec.2020.e00151","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00151","url":null,"abstract":"<div><p>L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type <em>Escherichia coli</em> for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from <em>Paracoccus aminovorans</em>. A xylose-induced T7 RNA polymerase-P<sub><em>T7</em></sub> promoter system was used to enhance and control <em>gmas</em> gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from <em>Corynebacterium glutamicum</em>. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from <em>C. glutamicum</em> was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from <em>Mannheimia succiniciproducens</em> was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 g/g glucose and 2.72 g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of <em>E. coli</em> for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00151","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91986221","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 : 2020-12-01DOI: 10.1016/j.mec.2020.e00132
Diego Tec-Campos , Cristal Zuñiga , Anurag Passi , John Del Toro , Juan D. Tibocha-Bonilla , Alejandro Zepeda , Michael J. Betenbaugh , Karsten Zengler
Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH3). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date.
Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.
固氮是微生物将分子氮转化为氨(NH3)等无机氮化合物的重要代谢过程。这些含氮化合物对生物地球化学循环和基本生物分子(即核酸、氨基酸和蛋白质)的合成至关重要。固氮杆菌是一种研究好氧固氮(重氮化)和产氢的细菌非光合模式生物。此外,重氮营养盐可以生产海藻酸盐和聚羟基丁酸盐(PHB)等生物聚合物,具有重要的工业应用。然而,许多代谢过程,如碳和氮代谢的分配,至今仍不清楚。基因组尺度代谢模型(m -模型)是在基因组尺度上揭示和优化代谢功能的可靠工具。m模型是包含有关基因、反应、代谢物及其关联的信息的数学表示。m -模型可以利用实验确定的约束条件模拟各种条件下的最优反应通量。在这里,我们报道了野生型细菌a . vinelandii DJ (iDT1278)的m模型的发展,该模型由2003种代谢物,2,469种反应和1,278个基因组成。我们利用高通量表型和生理数据验证了该模型,测试了180个碳源和95个氮源。iDT1278在碳源和氮源条件下的生长精度分别达到89%和91%。这种综合的m模型将有助于理解与固氮、铵同化和有机氮生产有关的代谢过程。
{"title":"Modeling of nitrogen fixation and polymer production in the heterotrophic diazotroph Azotobacter vinelandii DJ","authors":"Diego Tec-Campos , Cristal Zuñiga , Anurag Passi , John Del Toro , Juan D. Tibocha-Bonilla , Alejandro Zepeda , Michael J. Betenbaugh , Karsten Zengler","doi":"10.1016/j.mec.2020.e00132","DOIUrl":"https://doi.org/10.1016/j.mec.2020.e00132","url":null,"abstract":"<div><p>Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH<sub>3</sub>). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. <em>Azotobacter vinelandii</em> is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in <em>A. vinelandii</em> remain unknown to date.</p><p>Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium <em>A. vinelandii</em> DJ (<em>i</em>DT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. <em>i</em>DT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2020-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2020.e00132","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92066538","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}