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.

scholarly journals A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Amy B. Banta ◽  
Amy L. Enright ◽  
Cheta Siletti ◽  
Jason M. Peters
Keyword(s):  
Gene Function ◽  
Zymomonas Mobilis ◽  
Strain Engineering ◽  
Essential Genes ◽  
Biofuel Production ◽  
Alcohol Tolerance ◽  
High Efficacy ◽  
Content Type ◽  
Crispr Interference ◽  
Function Discovery

ABSTRACT Zymomonas 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 those of 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 essential genes. 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, are 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. IMPORTANCE Biofuels 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 like 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 explore gene function in Z. mobilis. We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

scholarly journals The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content

2021 ◽  
Vol 87 (6) ◽  
Author(s):  
Katsuya Fuchino ◽  
Helena Chan ◽  
Ling Chin Hwang ◽  
Per Bruheim
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Cell Size ◽  
Dna Content ◽  
Bacterial Cell ◽  
Zymomonas Mobilis ◽  
Biofuel Production ◽  
Public Attention ◽  
Content Type ◽  
Cell Organization

ABSTRACT The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilis. IMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker’s yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

scholarly journals Acid pretreatment and fractionation of source-separated organic waste for lignocellulosic saccharification

2021 ◽  
Author(s):  
Mandana Ehsanipour
Keyword(s):  
Organic Solvent ◽  
Zymomonas Mobilis ◽  
Biofuel Production ◽  
Acid Pretreatment ◽  
Cellulose Solvent ◽  
Continuous Bioreactor ◽  
Dilute Sulfuric Acid Pretreatment ◽  
Sulfuric Acid Pretreatment ◽  
Future Work ◽  
Hydrolysis Of

This study compared two acidic pretreatments on Source-Separated Organic (SSO) waste preprocessed by Aufbereitungs Technology and System thermal-screw, on the basis of fermentable sugars for bioethanol production. The result showed that the SSO contained on average 27% glucan, 5.4% xylan, 1.2% arabinan, 5.7% mannan and 1.2% galactan. Dilute sulfuric acid pretreatment (at 121°C and 16.2 psi) was insufficient to solubilize cellulose and hemicellulose and did not remove much of the lignin. Cellulose-solvent and Organic Solvent-based Lignocellulose Fractionation (COSLIF) (at 50°C and atmospheric pressure) generated high glucose yield (70%). Substituting ethanol for acetone as organic solvent increased the yield to 89.5%. Fermentation using Zymomonas mobilis 8b with this hydrolysate confirmed the pretreatment is promising for the SSO conversion. Amenability of the SSO for biofuel production is validated. Enzymatic hydrolysis of both pretreatments using Accellerase 1500 is preferred over Celluclast 1.5L due to higher activity. Future work includes design of an appropriate batch and/or continuous bioreactor, and further understanding of Zymomonas mobilis 8b.

Engineering of Zymomonas mobilis for Enhanced Biofuel Production

2021 ◽  
pp. 155-181
Author(s):  
Muneeba Khalid ◽  
Nasheen Rubab ◽  
Wajiha Afzal ◽  
Muhammad Irfan ◽  
Misbah Ghazanfar ◽  
...  
Keyword(s):  
Zymomonas Mobilis ◽  
Biofuel Production

scholarly journals Characterizing genetic diversity and creating novel gene pools in rice for trait dissection and gene function discovery

2010 ◽  
Author(s):  
Ramil Mauleon ◽  
Ramil Mauleon ◽  
Kenneth McNally ◽  
Hei Leung ◽  
Richard Bruskiewich ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gene Function ◽  
Gene Pools ◽  
Novel Gene ◽  
Function Discovery

scholarly journals Elucidation of Zymomonas mobilis physiology and stress responses by quantitative proteomics and transcriptomics

2014 ◽  
Vol 5 ◽  
Author(s):  
Shihui Yang ◽  
Chongle Pan ◽  
Gregory B. Hurst ◽  
Lezlee Dice ◽  
Brian H. Davison ◽  
...  
Keyword(s):  
Stress Responses ◽  
Quantitative Proteomics ◽  
Zymomonas Mobilis

scholarly journals Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans

2005 ◽  
Vol 73 (6) ◽  
pp. 3568-3576 ◽  
Author(s):  
Bing Wang ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Gene Function ◽  
Antisense Rna ◽  
Coenzyme A ◽  
Essential Gene ◽  
Essential Genes ◽  
Expression Vectors ◽  
Rna Expression ◽  
Gene Encoding ◽  
Antisense Dna

ABSTRACT In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.

scholarly journals VTR: an algorithm for identifying analogous contacts on protein structures and their complexes

2020 ◽  
Author(s):  
Vitor Pimentel ◽  
Diego Mariano ◽  
Letícia Xavier Silva Cantão ◽  
Luana Luiza Bastos ◽  
Pedro Fischer ◽  
...  
Keyword(s):  
Case Studies ◽  
Drug Targets ◽  
Rational Design ◽  
Structural Alignment ◽  
Protein Structures ◽  
Cell Receptor ◽  
Industrial Applications ◽  
Biofuel Production ◽  
Homologous Proteins ◽  
Related Proteins

Abstract Evolutionarily related proteins can present similar structures but very dissimilar sequences. Hence, understanding the role of the inter-residues contacts for the protein structure has been the target of many studies. Contacts comprise non-covalent interactions, which are essential to stabilize macromolecular structures such as proteins. Here we show VTR, a new method for the detection of analogous contacts in protein pairs. VTR performs structural alignment between proteins and detects interactions that occur in similar regions. To evaluate our tool, we proposed three case studies: (i) we compared a vertebrate myoglobin and a truncated invertebrate hemoglobin; (ii) analyzed interactions between the spike protein RBD of SARS-CoV-2 and the cell receptor ACE2; and (iii) compared a glucose-tolerant and a non-tolerant β-glucosidase enzyme used for biofuel production. The case studies demonstrate the potential of VTR for the understanding of functional similarities between distantly sequence-related proteins, as well as the exploration of important drug targets and rational design of enzymes for industrial applications. We envision VTR as a promising tool for understanding differences and similarities between homologous proteins with similar 3D structures but different sequences. VTR is available at http://bioinfo.dcc.ufmg.br/vtr.

scholarly journals Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate

2021 ◽  
Author(s):  
Magdalena M Felczak ◽  
Michaela A TerAvest
Keyword(s):  
Escherichia Coli ◽  
Zymomonas Mobilis ◽  
Biofuel Production ◽  
Growth Defect ◽  
Lignocellulosic Hydrolysate ◽  
Acetate Production ◽  
Bacterial Host ◽  
Acetaldehyde Dehydrogenase ◽  
Acetaldehyde Oxidation

Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase.

scholarly journals VTR: A Web Tool for Identifying Analogous Contacts on Protein Structures and Their Complexes

2021 ◽  
Vol 1 ◽  
Author(s):  
Vitor Pimentel ◽  
Diego Mariano ◽  
Letícia Xavier Silva Cantão ◽  
Luana Luiza Bastos ◽  
Pedro Fischer ◽  
...  
Keyword(s):  
Case Studies ◽  
Drug Targets ◽  
Rational Design ◽  
Structural Alignment ◽  
Protein Structures ◽  
Industrial Applications ◽  
Biofuel Production ◽  
Web Tool ◽  
Homologous Proteins ◽  
Related Proteins

Evolutionarily related proteins can present similar structures but very dissimilar sequences. Hence, understanding the role of the inter-residues contacts for the protein structure has been the target of many studies. Contacts comprise non-covalent interactions, which are essential to stabilize macromolecular structures such as proteins. Here we show VTR, a new method for the detection of analogous contacts in protein pairs. The VTR web tool performs structural alignment between proteins and detects interactions that occur in similar regions. To evaluate our tool, we proposed three case studies: we 1) compared vertebrate myoglobin and truncated invertebrate hemoglobin; 2) analyzed interactions between the spike protein RBD of SARS-CoV-2 and the cell receptor ACE2; and 3) compared a glucose-tolerant and a non-tolerant β-glucosidase enzyme used for biofuel production. The case studies demonstrate the potential of VTR for the understanding of functional similarities between distantly sequence-related proteins, as well as the exploration of important drug targets and rational design of enzymes for industrial applications. We envision VTR as a promising tool for understanding differences and similarities between homologous proteins with similar 3D structures but different sequences. VTR is available at http://bioinfo.dcc.ufmg.br/vtr.

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