scholarly journals Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

2021 ◽  
Author(s):  
Jin Luo ◽  
Emily A. McIntyre ◽  
Stacy R. Bedore ◽  
Ville Santala ◽  
Ellen L. Neidle ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Whole Genome ◽  
Acinetobacter Baylyi ◽  
Adaptive Laboratory Evolution ◽  
Natural Competence ◽  
Lignin Valorization ◽  
Laboratory Evolution ◽  
Recombination Efficiency ◽  
Novel Approach ◽  
Powerful Approach

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains evolved from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. Mutated DNA, added directly to growing cells, replaces homologous chromosomal regions to generate transformants that will become enriched if there is selective benefit. Thus, advantageous mutations can be rapidly identified. Here, the growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE , hcaK , and vanK , and a gene, ACIAD0482 , which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. Importance Microbial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Although adaptive laboratory evolution (ALE) is a powerful approach to develop more tolerant strains, it is typically laborious to identify the mechanisms underlying phenotypic improvement. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of this strain can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach for rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1-derived strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

scholarly journals Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

2021 ◽  
Author(s):  
Jin Luo ◽  
Emily A. McIntyre ◽  
Stacy R. Bedore ◽  
Ville Santala ◽  
Ellen L. Neidle ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Whole Genome ◽  
Acinetobacter Baylyi ◽  
Adaptive Laboratory Evolution ◽  
Natural Competence ◽  
Lignin Valorization ◽  
Laboratory Evolution ◽  
Recombination Efficiency ◽  
Novel Approach ◽  
Powerful Approach

AbstractAdaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains derived from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed both rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. The growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies.ImportanceMicrobial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Adaptive laboratory evolution is a powerful approach to develop more tolerant strains, but revealing the underlying mechanisms behind phenotypic improvement typically involves laborious processes. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of strain ADP1 can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach that allows rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1 strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

Novel approach of adaptive laboratory evolution: triggers defense molecules in Streptomyces sp. against targeted pathogen

RSC Advances ◽  
2016 ◽  
Vol 6 (98) ◽  
pp. 96250-96262 ◽  
Author(s):  
Sudarshan Singh Rathore ◽  
Vigneshwari Ramamurthy ◽  
Sally Allen ◽  
S. Selva Ganesan ◽  
Jayapradha Ramakrishnan
Keyword(s):  
Bioactive Molecules ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Streptomyces Sp ◽  
Novel Approach ◽  
Defense Molecules

Adaptive laboratory evolution by competition-based co-culture: triggers and enhance specific bioactive molecules against targeted pathogen.

scholarly journals Improving isobutanol productivity through adaptive laboratory evolution in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Aili Zhang ◽  
Yide Su ◽  
Jingzhi Li ◽  
Weiwei Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Metabolic Engineering ◽  
Glucose Concentration ◽  
Evolutionary Engineering ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Whole Genome Resequencing

Abstract Background: Isobutanol is an ideal second-generation biofuels due to its lower hygroscopicity, higher energy density and higher-octane value. However, isobutanol is toxic to production organisms. To improve isobutanol productivity, adaptive laboratory evolution method was carried out to improve the tolerance of Saccharomyces cerevisiae toward higher isobutanol and higher glucose concentration.Results: We evolved the laboratory strain of S. cerevisiae W303-1A by using EMS (ethyl methanesulfonate) mutagenesis followed by adaptive laboratory evolution. The evolved strain EMS39 with significant increase in growth rate and viability in media with higher isobutanol and higher glucose concentration was obtained. Then, metabolic engineering of the evolved strain EMS39 as a platform for isobutanol production were carried out. Delta integration method was used to over-express ILV3 gene and 2μ plasmids carrying ILV2, ILV5 and ARO10 were used to over-express ILV2, ILV5 and ARO10 genes in the evolved strain EMS39 and wild type W303-1A. And the resulting strains was designated as strain EMS39V2δV3V5A10 and strain W303-1AV2δV3V5A10, respectively. Our results shown that isobutanol titers of the evolved strain EMS39 increased by 30% compared to the control strain. And isobutanol productivity of strain EMS39V2δV3V5A10 increased by 32.4% compared to strain W303-1AV2δV3V5A10. Whole genome resequencing and analysis of site-directed mutagenesis of the evolved strain EMS39 have identified important mutations. In addition, RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from EMS39.Conclusions: With the aim of increase productivity of isobutanol in S. cerevisiae, improving tolerance toward higher isobutanol and higher glucose concentration via EMS mutagenesis followed by adaptive evolutionary engineering was conducted. An evolved strain EMS39 with significant increase in growth rate and viability had been obtained. And metabolic engineering of the evolved strain as a platform for isobutanol production was carried out. Furthermore, analysis of whole genome resequencing and transcriptome sequencing were also carried out.

scholarly journals Elucidating aromatic acid tolerance at low pH inSaccharomyces cerevisiaeusing adaptive laboratory evolution

2020 ◽  
Vol 117 (45) ◽  
pp. 27954-27961
Author(s):  
Rui Pereira ◽  
Elsayed T. Mohamed ◽  
Mohammad S. Radi ◽  
Markus J. Herrgård ◽  
Adam M. Feist ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Point Mutations ◽  
Acid Tolerance ◽  
Solubility Limit ◽  
Low Ph ◽  
Coumaric Acid ◽  
Aromatic Acids ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Cell Factories

Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generateSaccharomyces cerevisiaemutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80,ESBP6was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native toS. cerevisiae(Pdr12). Besides conferring tolerance to aromatic acids,ESBP6overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.

scholarly journals Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis

2021 ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
Benjamin Furtwängler ◽  
Matthijs Blommestijn ◽  
Adelė Kaltenytė ◽  
Jaap van der Poel ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Atp Synthase ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Constitutive Expression ◽  
Solvent Tolerance ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Trade Offs ◽  
Biobased Production

Pseudomonas putida S12 is inherently solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in the plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain. Whole genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% (vol/vol)). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β’, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. Transcriptomic analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, such as flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of solvent-tolerance trait in P. putida S12 lacking its megaplasmid. Importance: Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida, however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we begin to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida. This work sheds a light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

scholarly journals MICROEVOLUTION OF DESULFOVIBRIO VULGARIS CO-CULTURED WITH METHANOSARCINA BARKERI REVEALED BY GENOME RE-SEQUENCING AND SINGLE-CELL RT-QPCR ANALYSIS

2021 ◽  
Vol 80 (2) ◽  
pp. 109-124
Author(s):  
Zhenhua Qi ◽  
Xynyu Song ◽  
Zixi Chen
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Methanosarcina Barkeri ◽  
Integrative Approach ◽  
Desulfovibrio Vulgaris ◽  
Whole Genome ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Qpcr Analysis ◽  
Dual Cultures

An integrative approach of adaptive laboratory evolution, whole-genome sequencing and single-cell analysis was used to explore mechanisms related to establishment and maintenance of syntrophic interaction between sulfate-reducing Desulfovibrio vulgaris and methanogen Methanosarcina barkeri. Adaptive laboratory evolution of the D. vulgaris and M. barkeri dual-cultures under two different concentrations of electron donor lactate (38 mM and 50 mM) was conducted by propagating continuously for 50 transfers (~200 generations). Physiological analysis showed that, compared with the initial dual-cultures, the adapted dual-cultures (E38 and E50) have increased growth rates (1.1-fold and 1.2 -fold) and higher biomass yields (3.0-fold and 3.8-fold) on 38 mM and 50 mM lactate, respectively. Whole-genome re-sequencing of D. vulgaris in the adapted dual-cultures revealed 11 and 12 mutations in the D. vulgaris genomes of E38 and E50 dual-cultures, respectively, among which 4 mutations were found in both adapted dual-cultures. RT-qPCR analysis showed that the expression levels of 8 mutated genes were gradually up-regulated in D. vulgaris along with the evolution process. In addition, their heterogeneity was found decreased along with the evolution, as revealed by single-cell RT-qPCR analysis, reflecting adjustments of both gene expression and gene heterogeneity to the gradually established syntrophic relationship.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals Hydrogen-Free Deoxygenation of Bio-Oil Model Compounds over Sulfur-Free Polymer Supported Catalysts

2020 ◽  
Vol 7 (1) ◽  
pp. 29-36
Author(s):  
Antonina A. Stepacheva ◽  
Mariia E. Markova ◽  
Yury V. Lugovoy ◽  
Kirill V. Chalov ◽  
Mikhail G. Sulman ◽  
...  
Keyword(s):  
Supercritical Fluids ◽  
Aromatic Compounds ◽  
Supported Catalysts ◽  
Major Product ◽  
Hypercrosslinked Polystyrene ◽  
Model Compounds ◽  
Transportation Fuel ◽  
Novel Approach ◽  
Bio Oil ◽  
Hydrogen Donors

AbstractHydrotreatment of bio-oil oxygen compounds allows the final product to be effectively used as a liquid transportation fuel from biomass. Deoxygenation is considered to be one of the most promising ways for bio-oil upgrading. In the current work, we describe a novel approach for the deoxygenation of bio-oil model compounds (anisole, guaiacol) using supercritical fluids as both the solvent and hydrogen-donors. We estimated the possibility of the use of complex solvent consisting of non-polar n-hexane with low critical points (Tc = 234.5 ºC, Pc = 3.02 MPa) and propanol-2 used as H-donor. The experiments were performed without catalysts and in the presence of noble and transition metals hydrothermally deposited on the polymeric matrix of hypercrosslinked polystyrene (HPS). The experiments showed that the presence of 20 vol. % of propanol-2 in n-hexane results in the highest (up to 99%) conversion of model compounds. When the process was carried out without a catalyst, phenols were found to be a major product yielding up to 95 %. The use of Pd- and Co-containing catalyst yielded 90 % of aromatic compounds (benzene and toluene) while in the presence of Ru and Ni cyclohexane and methylcyclohexane (up to 98 %) were the main products.

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