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1.
Article in English | MEDLINE | ID: mdl-38925657

ABSTRACT

With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. ONE-SENTENCE SUMMARY: The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.


Subject(s)
Bacteria , Genomics , Bacteria/genetics , CRISPR-Cas Systems , Metabolic Engineering/methods , Industrial Microbiology , Gene Editing/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Genetic Engineering/methods
2.
Metab Eng ; 75: 78-90, 2023 01.
Article in English | MEDLINE | ID: mdl-36368470

ABSTRACT

Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.


Subject(s)
Cupriavidus necator , Cupriavidus necator/genetics , Cupriavidus necator/metabolism , Carbon Dioxide/metabolism , Operon , Carbon/metabolism , Formates/metabolism
3.
Transgenic Res ; 31(6): 661-676, 2022 12.
Article in English | MEDLINE | ID: mdl-36239844

ABSTRACT

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.


Subject(s)
Panicum , Transformation, Genetic , Panicum/genetics , Methionine/genetics , Agrobacterium tumefaciens/genetics , Transgenes , Plants, Genetically Modified/genetics , Plants, Genetically Modified/microbiology
4.
Metab Eng Commun ; 15: e00204, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36093381

ABSTRACT

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, ß-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the ßKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

6.
Biotechnol J ; 17(10): e2100673, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35766313

ABSTRACT

Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.


Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , DNA , Gene Editing/methods , Gene Targeting , Genome, Plant/genetics , Plant Breeding/methods , Plants/genetics , RNA, Guide, Kinetoplastida , RNA-Directed DNA Polymerase/genetics
7.
Synth Biol (Oxf) ; 7(1): ysac006, 2022.
Article in English | MEDLINE | ID: mdl-35734540

ABSTRACT

Saturation mutagenesis is a semi-rational approach for protein engineering where sites are saturated either entirely or partially to include amino acids of interest. We previously reported on a codon compression algorithm, where a set of minimal degenerate codons are selected according to user-defined parameters such as the target organism, type of saturation and usage levels. Here, we communicate an addition to our web tool that considers the distance between the wild-type codon and the library, depending on its purpose. These forms of restricted collections further reduce library size, lowering downstream screening efforts or, in turn, allowing more comprehensive saturation of multiple sites. The library design tool can be accessed via http://www.dynamcc.com/dynamcc_d/. Graphical Abstract.

8.
Biodes Res ; 2022: 9863496, 2022.
Article in English | MEDLINE | ID: mdl-37850147

ABSTRACT

Plants adapt to their changing environments by sensing and responding to physical, biological, and chemical stimuli. Due to their sessile lifestyles, plants experience a vast array of external stimuli and selectively perceive and respond to specific signals. By repurposing the logic circuitry and biological and molecular components used by plants in nature, genetically encoded plant-based biosensors (GEPBs) have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected. GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development. GEPBs are also useful for environmental monitoring, plant abiotic and biotic stress management, and accelerating design-build-test-learn cycles of plant bioengineering. With the advent of synthetic biology, biological and molecular components derived from alternate natural organisms (e.g., microbes) and/or de novo parts have been used to build GEPBs. In this review, we summarize the framework for engineering different types of GEPBs. We then highlight representative validated biological components for building plant-based biosensors, along with various applications of plant-based biosensors in basic and applied plant science research. Finally, we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.

9.
Metab Eng ; 67: 308-320, 2021 09.
Article in English | MEDLINE | ID: mdl-34245888

ABSTRACT

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.


Subject(s)
Escherichia coli , Systems Biology , Escherichia coli/genetics , Ethylenes , Laboratories , Metabolic Engineering , Pseudomonas syringae/genetics
10.
ACS Synth Biol ; 10(1): 19-28, 2021 01 15.
Article in English | MEDLINE | ID: mdl-33356165

ABSTRACT

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. In this study, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3-C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3-C4 alcohols by perturbing the transcriptional regulatory network.


Subject(s)
2-Propanol/pharmacology , Butanols/pharmacology , Gene Regulatory Networks/drug effects , Saccharomyces cerevisiae/genetics , Biofuels , Down-Regulation/drug effects , Drug Tolerance/genetics , Gene Library , Genome, Fungal , Glycolysis/drug effects , Glycolysis/genetics , Up-Regulation/drug effects
11.
Curr Opin Biotechnol ; 67: 7-14, 2021 02.
Article in English | MEDLINE | ID: mdl-33152605

ABSTRACT

Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Additionally, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.


Subject(s)
Gene Editing , Metabolic Engineering , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Genomics , Mutagenesis
12.
Nat Commun ; 11(1): 4050, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32792485

ABSTRACT

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli, with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Metabolic Engineering/methods , Synthetic Biology/methods , Gene Regulatory Networks/genetics , Gene Regulatory Networks/physiology
13.
Article in English | MEDLINE | ID: mdl-32719784

ABSTRACT

Biofuel production from renewable and sustainable resources is playing an increasingly important role within the fuel industry. Among biofuels, bioethanol has been most widely used as an additive for gasoline. Higher alcohols can be blended at a higher volume compared to ethanol and generate lower greenhouse gas (GHG) emissions without a need to change current fuel infrastructures. Thus, these fuels have the potential to replace fossil fuels in support of more environmentally friendly processes. This review summarizes the efforts to enhance bioalcohol production in engineered Escherichia coli over the last 5 years and analyzes the current challenges for increasing productivities for industrial applications.

14.
ACS Synth Biol ; 9(8): 2197-2202, 2020 08 21.
Article in English | MEDLINE | ID: mdl-32551581

ABSTRACT

Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.


Subject(s)
Saccharomyces cerevisiae/metabolism , Single-Chain Antibodies/metabolism , Antigen-Antibody Reactions , CRISPR-Cas Systems/genetics , Flow Cytometry , Gene Editing/methods , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Protein Engineering , Protein Stability , Saccharomyces cerevisiae/genetics , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics
15.
Molecules ; 25(9)2020 May 11.
Article in English | MEDLINE | ID: mdl-32403408

ABSTRACT

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.


Subject(s)
Aldose-Ketose Isomerases/chemistry , Directed Molecular Evolution/methods , Drug Resistance/genetics , Malaria/drug therapy , Mutagenesis , Aldose-Ketose Isomerases/antagonists & inhibitors , Aldose-Ketose Isomerases/metabolism , Anti-Bacterial Agents/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Fosfomycin/analogs & derivatives , Fosfomycin/pharmacology , Gene Library , Mutation , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
16.
Metab Eng Commun ; 10: e00116, 2020 Jun.
Article in English | MEDLINE | ID: mdl-31890588

ABSTRACT

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

17.
J Ind Microbiol Biotechnol ; 46(7): 993-1002, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30968274

ABSTRACT

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.


Subject(s)
Hydrogen/metabolism , Oxidoreductases/metabolism , Rhodopseudomonas/metabolism , Catalytic Domain , Gases , Oxidation-Reduction , Photosynthesis , Water
18.
Metab Eng ; 51: 50-58, 2019 01.
Article in English | MEDLINE | ID: mdl-30030154

ABSTRACT

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.


Subject(s)
Ethanol/metabolism , Ethanol/pharmacology , Metabolic Engineering/methods , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , CRISPR-Cas Systems , Fermentation , Gene Expression , Gene Library , Gene Regulatory Networks , Mutation , Plasmids/genetics , Saccharomyces cerevisiae/genetics
19.
ACS Synth Biol ; 7(12): 2824-2832, 2018 12 21.
Article in English | MEDLINE | ID: mdl-30462485

ABSTRACT

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.


Subject(s)
Aldose-Ketose Isomerases/genetics , Antimalarials/pharmacology , Drug Resistance/drug effects , Fosfomycin/analogs & derivatives , Aldose-Ketose Isomerases/metabolism , Antimalarials/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Escherichia coli/chemistry , Escherichia coli/metabolism , Fosfomycin/metabolism , Fosfomycin/pharmacology , Genetic Engineering/methods , Mutation , Plasmids/genetics , Plasmids/metabolism , Plasmodium falciparum/drug effects
20.
Biotechnol J ; 13(9): e1700586, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29917318

ABSTRACT

In recent years CRISPR-Cas technologies have revolutionized microbial engineering approaches. Genome editing and non-editing applications of various CRISPR-Cas systems have expanded the throughput and scale of engineering efforts, as well as opened up new avenues for manipulating genomes of non-model organisms. As we expand the range of organisms used for biotechnological applications, we need to develop better, more versatile tools for manipulation of these systems. Here the authors summarize the current advances in microbial gene editing using CRISPR-Cas based tools and highlight state-of-the-art methods for high-throughput, efficient genome-scale engineering in model organisms Escherichia coli and Saccharomyces cerevisiae. The authors also review non-editing CRISPR-Cas applications available for gene expression manipulation, epigenetic remodeling, RNA editing, labeling, and synthetic gene circuit design. Finally, the authors point out the areas of research that need further development in order to expand the range of applications and increase the utility of these new methods.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Genome, Microbial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Phenotype , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
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