scholarly journals QTL-guided metabolic engineering of a complex trait

2016 ◽  
Author(s):  
Matthew J. Maurer ◽  
Lawrence Sutardja ◽  
Dominic Pinel ◽  
Stefan Bauer ◽  
Amanda L. Muehlbauer ◽  
...  
Keyword(s):  
Rational Design ◽  
Complex Trait ◽  
Integrated Approach ◽  
Strain Engineering ◽  
Host Factors ◽  
Miscanthus X Giganteus ◽  
Complex Phenotypes ◽  
Lignocellulosic Feedstocks ◽  
Reciprocal Hemizygosity Analysis ◽  
S288c Strain

AbstractEngineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness ofSaccharomyces cerevisiaeduring fermentation of aMiscanthus x giganteusplant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this “genetic blueprint”, and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait inS. cerevisiae, as well as other organisms.

scholarly journals Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

2009 ◽  
Vol 75 (9) ◽  
pp. 2705-2711 ◽  
Author(s):  
Daniel Klein-Marcuschamer ◽  
Christine Nicole S. Santos ◽  
Huimin Yu ◽  
Gregory Stephanopoulos
Keyword(s):  
Rna Polymerase ◽  
Underlying Mechanism ◽  
Strain Engineering ◽  
Alpha Subunit ◽  
Physiological Changes ◽  
Core Enzyme ◽  
Complex Phenotypes ◽  
Bacterial Rna ◽  
Tolerant Mutant ◽  
Solvent Tolerant

ABSTRACT Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, l-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

scholarly journals Strain evolution and novel downstream processing with integrated catalysis enable highly efficient co-production of 1,3-Propanediol and organic acid esters from crude glycerol

2021 ◽  
Author(s):  
Chijian Zhang ◽  
Shubhang Sharma ◽  
Chengwei Ma ◽  
An-Ping Zeng
Keyword(s):  
Crude Glycerol ◽  
Methyl Esters ◽  
Integrated Approach ◽  
Downstream Processing ◽  
Strain Engineering ◽  
Raw Material ◽  
Evolution System ◽  
Microbial Conversion ◽  
Simultaneous Production ◽  
Heterogeneous Catalytic

Bioconversion process with a single target product often lacks economic competitiveness owing to incomplete use of raw material and high costs of downstream processing (DSP). Here, we show with the microbial conversion of crude glycerol that an integrated strain engineering and catalytic conversion of the so-called byproducts can greatly improve DSP and the process economy. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72 and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The co-production of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for a complete use of the raw material with the simultaneous production of three appealing products which greatly improve the process economy and ecology.

scholarly journals Determination of the Kinetics and Thermodynamic Parameters of Lignocellulosic Biomass Subjected to the Torrefaction Process

Materials ◽  
2021 ◽  
Vol 14 (24) ◽  
pp. 7877
Author(s):  
Maja Ivanovski ◽  
Aleksandra Petrovic ◽  
Irena Ban ◽  
Darko Goricanec ◽  
Danijela Urbancl
Keyword(s):  
Carbon Content ◽  
Isoconversional Methods ◽  
Conversion Degree ◽  
Heating Rates ◽  
Miscanthus X Giganteus ◽  
Main Method ◽  
Air Atmosphere ◽  
Solid Biofuels ◽  
Lignocellulosic Feedstocks

The torrefaction process upgrades biomass characteristics and produces solid biofuels that are coal-like in their properties. Kinetics analysis is important for the determination of the appropriate torrefaction condition to obtain the best utilization possible. In this study, the kinetics (Friedman (FR) and Kissinger–Akahira–Sunose (KAS) isoconversional methods of two final products of lignocellulosic feedstocks, miscanthus (Miscanthus x giganteus) and hops waste (Humulus Lupulus), were studied under different heating rates (10, 15, and 20 °C/min) using thermogravimetry (TGA) under air atmosphere as the main method to investigate. The results of proximate and ultimate analysis showed an increase in HHV values, carbon content, and fixed carbon content, followed by a decrease in the VM and O/C ratios for both torrefied biomasses, respectively. FTIR spectra confirmed the chemical changes during the torrefaction process, and they corresponded to the TGA results. The average Eα for torrefied miscanthus increased with the conversion degree for both models (25–254 kJ/mol for FR and 47–239 kJ/mol for the KAS model). The same trend was noticed for the torrefied hops waste samples; the values were within the range of 14–224 kJ/mol and 60–221 kJ/mol for the FR and KAS models, respectively. Overall, the Ea values for the torrefied biomass were much higher than for raw biomass, which was due to the different compositions of the torrefied material. Therefore, it can be concluded that both torrefied products can be used as a potential biofuel source.

scholarly journals Integrated strain- and process design enable production of 220 g L−1 itaconic acid with Ustilago maydis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Hamed Hosseinpour Tehrani ◽  
Johanna Becker ◽  
Isabel Bator ◽  
Katharina Saur ◽  
Svenja Meyer ◽  
...  
Keyword(s):  
Itaconic Acid ◽  
Acid Production ◽  
Process Development ◽  
Ustilago Maydis ◽  
Integrated Approach ◽  
Strain Engineering ◽  
Total Acid ◽  
Regulatory Cascade ◽  
Wide Range ◽  
Efficient Fermentation

Abstract Background Itaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production. Results In this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus. Conclusion In this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

scholarly journals FORECAST EVALUATION OF HEAT PROTECTION AND MECHANICAL PROPERTIES OF INSULATING CONSTRUCTION CERAMIC MATERIALS

2021 ◽  
pp. 68-74
Author(s):  
Liudmyla Pavlivna Shchukina ◽  
Yaroslav Olegovych Halushka ◽  
Larysa Oleksanrivna Yashchenko ◽  
Stanislav Leonidovych Lihezin
Keyword(s):  
Mechanical Properties ◽  
Thermal Conductivity ◽  
Rational Design ◽  
Structural Model ◽  
Ceramic Matrix ◽  
Integrated Approach ◽  
Ceramic Materials ◽  
Operating Conditions ◽  
Construction Ceramic ◽  
Heat Shielding

An integrated approach to determine the rational design of wall ceramic products based on modeling their behavior under operating conditions is proposed. This approach was used in the development of technology for heat–efficient insulating construction ceramic materials for energy–saving construction. For two models of porous–hollow ceramic products with a porous frame (40 % of voids) and a dense frame (60 % of voids), a predictive assessment of their heat–shielding and mechanical properties was carried out. Calculations of the equivalent coefficient of thermal conductivity of models based on Fourier’s law established that with a decrease in the voidness of products with a porous wall, the coefficient of their thermal conductivity decreases by 12 %, which improves the heat–shielding properties. Based on the results of computer simulation of the behavior of models under the influence of static power loads, it was determined that porosity of the ceramic framework of products leads to degradation of mechanical strength almost proportionally to a decrease in voidness. The stress–strain state of 3D models of ceramic structures with different pore geometry (spherical, globular, ellipsoidal) is analyzed and it is shown that stresses are concentrated in the contact zones of a ceramic matrix with pores. It is shown that the most durable is the structural model with spherical pores. The expediency of organizing such a structure, the need to strengthen the ceramic matrix of materials and zones surrounding the pores, as the most vulnerable structural sites, is shown. The results of predictive calculations have been experimentally confirmed in the development of technology for structural and heat–insulating composite–type ceramic materials based on low–melting loam and ash microspheres, which provide a given structural picture of the ceramic material.

scholarly journals Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

2020 ◽  
Vol 8 (10) ◽  
pp. 1466
Author(s):  
Andrea Colarusso ◽  
Concetta Lauro ◽  
Marzia Calvanese ◽  
Ermenegilda Parrilli ◽  
Maria Luisa Tutino
Keyword(s):  
Recombinant Expression ◽  
Integrated Approach ◽  
Strain Engineering ◽  
Cell Factory ◽  
Lactose Permease ◽  
Wild Type ◽  
Iptg Induction ◽  
Pseudoalteromonas Haloplanktis ◽  
Pseudoalteromonas Haloplanktis Tac125 ◽  
In The Wild

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

scholarly journals An Integrated Approach for the Rational Design of Nanovectors for Biomedical Imaging and Therapy

2010 ◽  
pp. 31-64 ◽  
Author(s):  
Biana Godin ◽  
Wouter H. P. Driessen ◽  
Bettina Proneth ◽  
Sei-Young Lee ◽  
Srimeenakshi Srinivasan ◽  
...  
Keyword(s):  
Biomedical Imaging ◽  
Rational Design ◽  
Integrated Approach

Metabolic engineering of glycoprotein biosynthesis in bacteria

2018 ◽  
Vol 2 (3) ◽  
pp. 419-432 ◽  
Author(s):  
Aravind Natarajan ◽  
Thapakorn Jaroentomeechai ◽  
Mingji Li ◽  
Cameron J. Glasscock ◽  
Matthew P. DeLisa
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Campylobacter Jejuni ◽  
Rational Design ◽  
Strain Engineering ◽  
Protein Glycosylation ◽  
Microbial Cell ◽  
Evolutionary Engineering ◽  
Escherichia Coli Cells ◽  
Genome Wide

The demonstration more than a decade ago that glycoproteins could be produced in Escherichia coli cells equipped with the N-linked protein glycosylation machinery from Campylobacter jejuni opened the door to using simple bacteria for the expression and engineering of complex glycoproteins. Since that time, metabolic engineering has played an increasingly important role in developing and optimizing microbial cell glyco-factories for the production of diverse glycoproteins and other glycoconjugates. It is becoming clear that future progress in creating efficient glycoprotein expression platforms in bacteria will depend on the adoption of advanced strain engineering strategies such as rational design and assembly of orthogonal glycosylation pathways, genome-wide identification of metabolic engineering targets, and evolutionary engineering of pathway performance. Here, we highlight recent advances in the deployment of metabolic engineering tools and strategies to develop microbial cell glyco-factories for the production of high-value glycoprotein targets with applications in research and medicine.

scholarly journals A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

2020 ◽  
Author(s):  
Amy B. Banta ◽  
Amy L. Enright ◽  
Cheta Siletti ◽  
Jason M. Peters
Keyword(s):  
Gene Function ◽  
Stress Responses ◽  
Zymomonas Mobilis ◽  
Rational Design ◽  
Strain Engineering ◽  
Essential Genes ◽  
Biofuel Production ◽  
Alcohol Tolerance ◽  
High Efficacy ◽  
Function Discovery

ABSTRACTZymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essentials. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes, or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.IMPORTANCEBiofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next generation biofuels such as isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to characterize gene function in Z. mobilis. We identify genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to alcohol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

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