Pub Date : 2022-12-01DOI: 10.1016/j.mec.2022.e00208
Danielle Bever , Ian Wheeldon , Nancy Da Silva
The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.
{"title":"RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus","authors":"Danielle Bever , Ian Wheeldon , Nancy Da Silva","doi":"10.1016/j.mec.2022.e00208","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00208","url":null,"abstract":"<div><p>The thermotolerant yeast <em>Kluyveromyces marxianus</em> has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of <em>K. marxianus</em>, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional <em>K. marxianus</em> strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient <em>K. marxianus</em> strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in <em>K. marxianus</em> using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00208"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000177/pdfft?md5=f6be45d36815b9de356945d06bf05598&pid=1-s2.0-S2214030122000177-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91591437","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 : 2022-12-01DOI: 10.1016/j.mec.2022.e00212
Emil Ljungqvist, Martin Gustavsson
Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, an important part of the metabolic engineering pipeline, of the fast-growing thermophile Geobacillus sp. LC300. The model (iGEL604) contains 604 genes, 1249 reactions and 1311 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. The growth phenotype is analyzed by batch cultivations on two carbon sources, further closing balances in carbon and degree-of-reduction. The predictive ability of the model is benchmarked against experimentally determined growth characteristics and internal flux distributions, showing high similarity to experimental phenotypes.
{"title":"Genome-scale reconstruction and metabolic modelling of the fast-growing thermophile Geobacillus sp. LC300","authors":"Emil Ljungqvist, Martin Gustavsson","doi":"10.1016/j.mec.2022.e00212","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00212","url":null,"abstract":"<div><p>Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, an important part of the metabolic engineering pipeline, of the fast-growing thermophile <em>Geobacillus</em> sp. LC300. The model (iGEL604) contains 604 genes, 1249 reactions and 1311 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. The growth phenotype is analyzed by batch cultivations on two carbon sources, further closing balances in carbon and degree-of-reduction. The predictive ability of the model is benchmarked against experimentally determined growth characteristics and internal flux distributions, showing high similarity to experimental phenotypes.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00212"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000219/pdfft?md5=084bfef2d5e8e9c0bac22632181a9aff&pid=1-s2.0-S2214030122000219-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91640474","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}
Metabolic engineering involves the manipulation of microbes to produce desirable compounds through genetic engineering or synthetic biology approaches. Metabolomics involves the quantitation of intracellular and extracellular metabolites, where mass spectrometry and nuclear magnetic resonance based analytical instrumentation are often used. Here, the experimental designs, sample preparations, metabolite quenching and extraction are essential to the quantitative metabolomics workflow. The resultant metabolomics data can then be used with computational modelling approaches, such as kinetic and constraint-based modelling, to better understand underlying mechanisms and bottlenecks in the synthesis of desired compounds, thereby accelerating research through systems metabolic engineering. Constraint-based models, such as genome scale models, have been used successfully to enhance the yield of desired compounds from engineered microbes, however, unlike kinetic or dynamic models, constraint-based models do not incorporate regulatory effects. Nevertheless, the lack of time-series metabolomic data generation has hindered the usefulness of dynamic models till today. In this review, we show that improvements in automation, dynamic real-time analysis and high throughput workflows can drive the generation of more quality data for dynamic models through time-series metabolomics data generation. Spatial metabolomics also has the potential to be used as a complementary approach to conventional metabolomics, as it provides information on the localization of metabolites. However, more effort must be undertaken to identify metabolites from spatial metabolomics data derived through imaging mass spectrometry, where machine learning approaches could prove useful. On the other hand, single-cell metabolomics has also seen rapid growth, where understanding cell-cell heterogeneity can provide more insights into efficient metabolic engineering of microbes. Moving forward, with potential improvements in automation, dynamic real-time analysis, high throughput workflows, and spatial metabolomics, more data can be produced and studied using machine learning algorithms, in conjunction with dynamic models, to generate qualitative and quantitative predictions to advance metabolic engineering efforts.
{"title":"Metabolomics and modelling approaches for systems metabolic engineering","authors":"Jasmeet Kaur Khanijou , Hanna Kulyk , Cécilia Bergès , Leng Wei Khoo , Pnelope Ng , Hock Chuan Yeo , Mohamed Helmy , Floriant Bellvert , Wee Chew , Kumar Selvarajoo","doi":"10.1016/j.mec.2022.e00209","DOIUrl":"10.1016/j.mec.2022.e00209","url":null,"abstract":"<div><p>Metabolic engineering involves the manipulation of microbes to produce desirable compounds through genetic engineering or synthetic biology approaches. Metabolomics involves the quantitation of intracellular and extracellular metabolites, where mass spectrometry and nuclear magnetic resonance based analytical instrumentation are often used. Here, the experimental designs, sample preparations, metabolite quenching and extraction are essential to the quantitative metabolomics workflow. The resultant metabolomics data can then be used with computational modelling approaches, such as kinetic and constraint-based modelling, to better understand underlying mechanisms and bottlenecks in the synthesis of desired compounds, thereby accelerating research through systems metabolic engineering. Constraint-based models, such as genome scale models, have been used successfully to enhance the yield of desired compounds from engineered microbes, however, unlike kinetic or dynamic models, constraint-based models do not incorporate regulatory effects. Nevertheless, the lack of time-series metabolomic data generation has hindered the usefulness of dynamic models till today. In this review, we show that improvements in automation, dynamic real-time analysis and high throughput workflows can drive the generation of more quality data for dynamic models through time-series metabolomics data generation. Spatial metabolomics also has the potential to be used as a complementary approach to conventional metabolomics, as it provides information on the localization of metabolites. However, more effort must be undertaken to identify metabolites from spatial metabolomics data derived through imaging mass spectrometry, where machine learning approaches could prove useful. On the other hand, single-cell metabolomics has also seen rapid growth, where understanding cell-cell heterogeneity can provide more insights into efficient metabolic engineering of microbes. Moving forward, with potential improvements in automation, dynamic real-time analysis, high throughput workflows, and spatial metabolomics, more data can be produced and studied using machine learning algorithms, in conjunction with dynamic models, to generate qualitative and quantitative predictions to advance metabolic engineering efforts.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00209"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/2e/c7/main.PMC9587336.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40679625","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 : 2022-12-01DOI: 10.1016/j.mec.2022.e00210
Mamta Gupta , Matthew Wong , Kamran Jawed , Kamil Gedeon , Hannah Barrett , Marcelo Bassalo , Clifford Morrison , Danish Eqbal , Syed Shams Yazdani , Ryan T. Gill , Jiaqi Huang , Marc Douaisi , Jonathan Dordick , Georges Belfort , Mattheos A.G. Koffas
The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L−1 h−1, and titers of 5.6 and 1.78 g L−1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.
在大肠杆菌中生产生物燃料异丁醇面临着酒精毒性、产品抑制、产品回收和长期工业可行性的限制。在这里,我们展示了一种结合体内和体外代谢工程来生产异丁醇的方法。α-酮异戊酸(KIV)的体内生成是通过CRISPR介导的大肠杆菌双胞设计(BCD)中KIV通路的整合和CRISPRi技术对竞争缬氨酸通路的抑制来实现的。随后用2-酮酸脱羧酶(KIVD)和醇脱氢酶(ADH)工程酶进行体外异丁醇转化。对于KIV的体内生产和随后的体外异丁醇生产,这种两步连续方法的产率分别为56%和93%,产率分别为0.62和0.074 g L−1 h−1,滴度分别为5.6和1.78 g L−1。因此,这种组合的生物合成系统可以作为一种模块化的方法来生产重要的代谢物,如异丁醇,而不受体内使用统一生物过程生产的限制。
{"title":"Isobutanol production by combined in vivo and in vitro metabolic engineering","authors":"Mamta Gupta , Matthew Wong , Kamran Jawed , Kamil Gedeon , Hannah Barrett , Marcelo Bassalo , Clifford Morrison , Danish Eqbal , Syed Shams Yazdani , Ryan T. Gill , Jiaqi Huang , Marc Douaisi , Jonathan Dordick , Georges Belfort , Mattheos A.G. Koffas","doi":"10.1016/j.mec.2022.e00210","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00210","url":null,"abstract":"<div><p>The production of the biofuel, isobutanol, in <em>E. coli</em> faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both <em>in vivo</em> with <em>in vitro</em> metabolic engineering to produce isobutanol. The <em>in vivo</em> production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in <em>E. coli</em> and inhibition of competitive valine pathway using CRISPRi technology. The subsequent <em>in vitro</em> conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the <em>in vivo</em> production of KIV and subsequent <em>in vitro</em> production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L<sup>−1</sup> h<sup>−1</sup>, and titers of 5.6 and 1.78 g L<sup>−1</sup>, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with <em>in vivo</em> production using a consolidated bioprocess.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00210"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000190/pdfft?md5=b291a3d0e60096f33c80f32a13d96aa2&pid=1-s2.0-S2214030122000190-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91640475","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}
Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.
{"title":"Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species","authors":"Annie Yap , Irene Glarcher , Matthias Misslinger, Hubertus Haas","doi":"10.1016/j.mec.2022.e00214","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00214","url":null,"abstract":"<div><p>Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The <em>xylP</em> promoter (p<em>xylP</em>) from <em>Penicillium chrysogenum</em> was demonstrated to function in various mold species including <em>Aspergillus fumigatus</em>. p<em>xylP</em> allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered p<em>xylP</em> in <em>A. fumigatus</em> to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, p<em>xylP</em> activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of p<em>xylP</em> to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp p<em>xylP</em> was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of p<em>xylP</em>. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of p<em>xylP</em>. Taken together, this study identified functional elements of p<em>xylP</em> and opened new methodological opportunities for its application.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00214"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000232/pdfft?md5=43a616ce871d93dcd430b7cbcb1a7779&pid=1-s2.0-S2214030122000232-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89988962","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}
Hemicellulose is the second most abundant carbohydrate in lignocellulosic biomass and has extensive applications. In conventional biomass refinery, hemicellulose is easily converted to unwanted by-products in pretreatment and therefore can't be fully utilized. The present study aims to summarize the most recent development of lignocellulosic polysaccharide degradation and fully convert it to value-added bioproducts through microbial and enzymatic catalysis. Firstly, bioprocess and microbial metabolic engineering for enhanced utilization of lignocellulosic carbohydrates were discussed. The bioprocess for degradation and conversion of natural lignocellulose to monosaccharides and organic acids using anaerobic thermophilic bacteria and thermostable glycoside hydrolases were summarized. Xylose transmembrane transporting systems in natural microorganisms and the latest strategies for promoting the transporting capacity by metabolic engineering were summarized. The carbon catabolite repression effect restricting xylose utilization in microorganisms, and metabolic engineering strategies developed for co-utilization of glucose and xylose were discussed. Secondly, the metabolic pathways of xylose catabolism in microorganisms were comparatively analyzed. Microbial metabolic engineering for converting xylose to value-added bioproducts based on redox pathways, non-redox pathways, pentose phosphate pathway, and improving inhibitors resistance were summarized. Thirdly, strategies for degrading lignocellulosic polysaccharides and fully converting hemicellulose to value-added bioproducts through microbial metabolic engineering were proposed.
{"title":"Biosynthesis of value-added bioproducts from hemicellulose of biomass through microbial metabolic engineering","authors":"Biao Geng , Xiaojing Jia , Xiaowei Peng , Yejun Han","doi":"10.1016/j.mec.2022.e00211","DOIUrl":"10.1016/j.mec.2022.e00211","url":null,"abstract":"<div><p>Hemicellulose is the second most abundant carbohydrate in lignocellulosic biomass and has extensive applications. In conventional biomass refinery, hemicellulose is easily converted to unwanted by-products in pretreatment and therefore can't be fully utilized. The present study aims to summarize the most recent development of lignocellulosic polysaccharide degradation and fully convert it to value-added bioproducts through microbial and enzymatic catalysis. Firstly, bioprocess and microbial metabolic engineering for enhanced utilization of lignocellulosic carbohydrates were discussed. The bioprocess for degradation and conversion of natural lignocellulose to monosaccharides and organic acids using anaerobic thermophilic bacteria and thermostable glycoside hydrolases were summarized. Xylose transmembrane transporting systems in natural microorganisms and the latest strategies for promoting the transporting capacity by metabolic engineering were summarized. The carbon catabolite repression effect restricting xylose utilization in microorganisms, and metabolic engineering strategies developed for co-utilization of glucose and xylose were discussed. Secondly, the metabolic pathways of xylose catabolism in microorganisms were comparatively analyzed. Microbial metabolic engineering for converting xylose to value-added bioproducts based on redox pathways, non-redox pathways, pentose phosphate pathway, and improving inhibitors resistance were summarized. Thirdly, strategies for degrading lignocellulosic polysaccharides and fully converting hemicellulose to value-added bioproducts through microbial metabolic engineering were proposed.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00211"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9597109/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40443804","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 : 2022-12-01DOI: 10.1016/j.mec.2022.e00202
Vaishnavi Sivapuratharasan , Christoph Lenzen , Carina Michel , Anantha Barathi Muthukrishnan , Guhan Jayaraman , Lars M. Blank
Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.
{"title":"Metabolic engineering of Pseudomonas taiwanensis VLB120 for rhamnolipid biosynthesis from biomass-derived aromatics","authors":"Vaishnavi Sivapuratharasan , Christoph Lenzen , Carina Michel , Anantha Barathi Muthukrishnan , Guhan Jayaraman , Lars M. Blank","doi":"10.1016/j.mec.2022.e00202","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00202","url":null,"abstract":"<div><p>Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of <em>Pseudomonas taiwanensis</em> VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was <em>actP</em>, coding for a cation/acetate symporter, and the other gene was <em>paaA</em> coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmol<sub>rhl</sub>/Cmol<sub>4-coumarate</sub>) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmol<sub>rhl</sub>/Cmol<sub>ferulate</sub>), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00202"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000116/pdfft?md5=29575c540fc6b7e0e6bf633a82266d60&pid=1-s2.0-S2214030122000116-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89989006","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 : 2022-06-01DOI: 10.1016/j.mec.2021.e00191
Brandi Brown , Cheryl Immethun , Adil Alsiyabi , Dianna Long , Mark Wilkins , Rajib Saha
Rhodopseudomonas palustris CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by R. palustris. The Phap1 phasin (phaP1) from the PHB-producing model bacterium Cupriavidus necator H16 was expressed in R. palustris with the aim of overproducing PHBV from the lignin breakdown product p-coumarate by fostering smaller and more abundant granules. Expression of phaP1 yielded PHBV production from R. palustris aerobically (0.7 g/L), which does not occur in the wild-type strain, and led to a significantly higher PHBV titer than wild-type anaerobic production (0.41 g/L). The 3HV fractions were also significantly increased under both anaerobic and aerobic conditions, which boosts thermomechanical properties and potential for application. Thus, heterologous phasin expression in R. palustris provides flexibility for industrial processing and could foster compositional changes in copolymers with better thermomechanical properties compared to PHB alone.
{"title":"Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass","authors":"Brandi Brown , Cheryl Immethun , Adil Alsiyabi , Dianna Long , Mark Wilkins , Rajib Saha","doi":"10.1016/j.mec.2021.e00191","DOIUrl":"https://doi.org/10.1016/j.mec.2021.e00191","url":null,"abstract":"<div><p><em>Rhodopseudomonas palustris</em> CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by <em>R. palustris.</em> The Phap1 phasin (<em>phaP1</em>) from the PHB-producing model bacterium <em>Cupriavidus necator</em> H16 was expressed in <em>R. palustris</em> with the aim of overproducing PHBV from the lignin breakdown product <em>p-</em>coumarate by fostering smaller and more abundant granules. Expression of <em>phaP1</em> yielded PHBV production from <em>R. palustris</em> aerobically (0.7 g/L), which does not occur in the wild-type strain, and led to a significantly higher PHBV titer than wild-type anaerobic production (0.41 g/L). The 3HV fractions were also significantly increased under both anaerobic and aerobic conditions, which boosts thermomechanical properties and potential for application. Thus, heterologous phasin expression in <em>R. palustris</em> provides flexibility for industrial processing and could foster compositional changes in copolymers with better thermomechanical properties compared to PHB alone.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"14 ","pages":"Article e00191"},"PeriodicalIF":5.2,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030121000316/pdfft?md5=ff323c34605bef32932c28358858dc3e&pid=1-s2.0-S2214030121000316-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92014806","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 : 2022-06-01DOI: 10.1016/j.mec.2022.e00194
Soledad Mora-Vásquez , Guillermo Gael Wells-Abascal , Claudia Espinosa-Leal , Guy A. Cardineau , Silverio García-Lara
Plants are a rich source of bioactive compounds, many of which have been exploited for cosmetic, nutritional, and medicinal purposes. Through the characterization of metabolic pathways, as well as the mechanisms responsible for the accumulation of secondary metabolites, researchers have been able to increase the production of bioactive compounds in different plant species for research and commercial applications. The intent of the current review is to describe the metabolic engineering methods that have been used to transform in vitro or field-grown medicinal plants over the last decade and to identify the most effective approaches to increase the production of alkaloids. The articles summarized were categorized into six groups: endogenous enzyme overexpression, foreign enzyme overexpression, transcription factor overexpression, gene silencing, genome editing, and co-overexpression. We conclude that, because of the complex and multi-step nature of biosynthetic pathways, the approach that has been most commonly used to increase the biosynthesis of alkaloids, and the most effective in terms of fold increase, is the co-overexpression of two or more rate-limiting enzymes followed by the manipulation of regulatory genes.
{"title":"Application of metabolic engineering to enhance the content of alkaloids in medicinal plants","authors":"Soledad Mora-Vásquez , Guillermo Gael Wells-Abascal , Claudia Espinosa-Leal , Guy A. Cardineau , Silverio García-Lara","doi":"10.1016/j.mec.2022.e00194","DOIUrl":"10.1016/j.mec.2022.e00194","url":null,"abstract":"<div><p>Plants are a rich source of bioactive compounds, many of which have been exploited for cosmetic, nutritional, and medicinal purposes. Through the characterization of metabolic pathways, as well as the mechanisms responsible for the accumulation of secondary metabolites, researchers have been able to increase the production of bioactive compounds in different plant species for research and commercial applications. The intent of the current review is to describe the metabolic engineering methods that have been used to transform in vitro or field-grown medicinal plants over the last decade and to identify the most effective approaches to increase the production of alkaloids. The articles summarized were categorized into six groups: endogenous enzyme overexpression, foreign enzyme overexpression, transcription factor overexpression, gene silencing, genome editing, and co-overexpression. We conclude that, because of the complex and multi-step nature of biosynthetic pathways, the approach that has been most commonly used to increase the biosynthesis of alkaloids, and the most effective in terms of fold increase, is the co-overexpression of two or more rate-limiting enzymes followed by the manipulation of regulatory genes.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"14 ","pages":"Article e00194"},"PeriodicalIF":5.2,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8881666/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46036667","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 : 2022-06-01DOI: 10.1016/j.mec.2022.e00199
Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä
The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation in vivo at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (ldhA) from Leuconostoc mesenteroides to control D-lactic acid formation in yeast Saccharomyces cerevisiae. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the ldhA gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with ldhA expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation in vivo, and D-lactic acid content in the poly(D-lactate-co-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% in vivo in S. cerevisiae. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in S. cerevisiae the PHA synthase PhaC1437Ps6-19 produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.
{"title":"Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system","authors":"Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä","doi":"10.1016/j.mec.2022.e00199","DOIUrl":"10.1016/j.mec.2022.e00199","url":null,"abstract":"<div><p>The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation <em>in vivo</em> at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (<em>ldhA</em>) from <em>Leuconostoc mesenteroides</em> to control D-lactic acid formation in yeast <em>Saccharomyces cerevisiae</em>. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the <em>ldhA</em> gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with <em>ldhA</em> expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation <em>in vivo</em>, and D-lactic acid content in the poly(D-lactate-<em>co</em>-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% <em>in vivo</em> in <em>S. cerevisiae</em>. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in <em>S. cerevisiae</em> the PHA synthase PhaC1437<sub>Ps6-19</sub> produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"14 ","pages":"Article e00199"},"PeriodicalIF":5.2,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000086/pdfft?md5=46cba499ea94cf885917415684ffaf00&pid=1-s2.0-S2214030122000086-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46506594","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}