scholarly journals Heterologous glycosyl hydrolase expression and cellular reprogramming resembling sucrose-induction enable Zymomonas mobilis growth on cellobiose

2019 ◽  
Author(s):  
Nagendra P. Kurumbang ◽  
Jessica M. Vera ◽  
Alexander S. Hebert ◽  
Joshua J. Coon ◽  
Robert Landick
Keyword(s):  
Zymomonas Mobilis ◽  
Plant Biomass ◽  
Caulobacter Crescentus ◽  
Glycosyl Hydrolase ◽  
Surface Display ◽  
Genetic Mutation ◽  
Cellular Reprogramming ◽  
Lag Phase ◽  
Renewable Biomass ◽  
Sucrose Induction

ABSTRACTPlant derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from Caulobacter crescentus and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and secretion of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 hours and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid secretion or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that enables sucrose secretion.

scholarly journals Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Jessica M. Vera ◽  
Indro Neil Ghosh ◽  
Yaoping Zhang ◽  
Alex S. Hebert ◽  
Joshua J. Coon ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Zymomonas Mobilis ◽  
Plant Biomass ◽  
Caulobacter Crescentus ◽  
Functional Characterization ◽  
Ribosome Profiling ◽  
Start Codon ◽  
Label Free ◽  
Content Type ◽  
Functional Sites

ABSTRACT Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5′ and 3′ ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5′ end. Our results revealed that Z. mobilis σA −35 and −10 promoter elements closely resemble canonical Escherichia coli −35 and −10 elements, with one notable exception: the Z. mobilis −10 element lacks the highly conserved −7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of −7 thymine in their −10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis. This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

scholarly journals Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0226235
Author(s):  
Nagendra P. Kurumbang ◽  
Jessica M. Vera ◽  
Alexander S. Hebert ◽  
Joshua J. Coon ◽  
Robert Landick
Keyword(s):  
Heterologous Expression ◽  
Zymomonas Mobilis ◽  
Glycosyl Hydrolase ◽  
Cellular Reprogramming

scholarly journals Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces

2011 ◽  
Vol 77 (11) ◽  
pp. 3793-3801 ◽  
Author(s):  
Robyn Peterson ◽  
Jasmine Grinyer ◽  
Helena Nevalainen
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Plant Biomass ◽  
Glycosyl Hydrolase ◽  
Tandem Mass ◽  
Glycosyl Hydrolase Family ◽  
Coprophilous Fungi ◽  
Content Type ◽  
Coprophilous Fungus ◽  
Hydrolase Family

ABSTRACTCoprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization–time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography–tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungusDoratomyces stemonitisC8 (EU551185) isolated from koala feces. As the genome ofD. stemonitishas not been sequenced, cross-species identification,de novosequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in theD. stemonitisC8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploitedT. reesei. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.

scholarly journals Improving mobilization of foreign DNA into Zymomonas mobilis ZM4 by removal of multiple restriction systems

2021 ◽  
Author(s):  
Piyush Behari Lal ◽  
Fritz Wells ◽  
Kevin S. Myers ◽  
Rajdeep Banerjee ◽  
Adam M. Guss ◽  
...  
Keyword(s):  
Zymomonas Mobilis ◽  
Plant Biomass ◽  
Dna Recognition ◽  
Type I ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Recognition Motif ◽  
Restriction System ◽  
System A ◽  
Foreign Dna

Zymomonas mobilis has emerged as a promising candidate for production of high value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr -like Type IV Restriction Modification (RM) system, a Type I-F CRISPR system, a chromosomal Type I RM ( hsdMS c ) and a previously uncharacterized Type I RM system, located on an endogenous plasmid ( hsdRMS p ). The DNA recognition motif of HsdRMS p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS c and hsdRMS p systems to the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS c and HsdRMS p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction - strains that we constructed, and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. Importance Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study we have developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identified a previously unknown Type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis .

scholarly journals HdaB: a novel and conserved DnaA-related protein that targets the RIDA process to stimulate replication initiation

2019 ◽  
Vol 48 (5) ◽  
pp. 2412-2423 ◽  
Author(s):  
Antonio Frandi ◽  
Justine Collier
Keyword(s):  
Stationary Phase ◽  
Caulobacter Crescentus ◽  
Replication Initiation ◽  
Lag Phase ◽  
Related Protein ◽  
Free Living ◽  
Changing Environments ◽  
A Cell ◽  
The Impact

Abstract Exquisite control of the DnaA initiator is critical to ensure that bacteria initiate chromosome replication in a cell cycle-coordinated manner. In many bacteria, the DnaA-related and replisome-associated Hda/HdaA protein interacts with DnaA to trigger the Regulatory Inactivation of DnaA (RIDA) and prevent over-initiation events. In the Caulobacter crescentus Alphaproteobacterium, the RIDA process also targets DnaA for its rapid proteolysis by Lon. The impact of the RIDA process on adaptation of bacteria to changing environments remains unexplored. Here, we identify a novel and conserved DnaA-related protein, named HdaB, and show that homologs from three different Alphaproteobacteria can inhibit the RIDA process, leading to over-initiation and cell death when expressed in actively growing C. crescentus cells. We further show that HdaB interacts with HdaA in vivo, most likely titrating HdaA away from DnaA. Strikingly, we find that HdaB accumulates mainly during stationary phase and that it shortens the lag phase upon exit from stationary phase. Altogether, these findings suggest that expression of hdaB during stationary phase prepares cells to restart the replication of their chromosome as soon as conditions improve, a situation often met by free-living or facultative intracellular Alphaproteobacteria.

scholarly journals Cut-Lengths of Perennial Ryegrass Leaf-Blades Influences In Vitro Fermentation by the Anaerobic Fungus Neocallimastix frontalis

2020 ◽  
Vol 8 (11) ◽  
pp. 1774
Author(s):  
Hugo R. Jimenez ◽  
Joan E. Edwards ◽  
Ruth Sanderson ◽  
Alison H. Kingston-Smith ◽  
Neil R. McEwan ◽  
...  
Keyword(s):  
Perennial Ryegrass ◽  
Leaf Blade ◽  
Plant Biomass ◽  
Gas Production ◽  
Lag Phase ◽  
Anaerobic Fungi ◽  
In Vitro Fermentation ◽  
Batch Cultures ◽  
Neocallimastix Frontalis

Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host’s ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results.

scholarly journals Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost

2011 ◽  
Vol 77 (7) ◽  
pp. 2282-2291 ◽  
Author(s):  
M. V. Sizova ◽  
J. A. Izquierdo ◽  
N. S. Panikov ◽  
L. R. Lynd
Keyword(s):  
Enrichment Culture ◽  
Cellulose Degradation ◽  
Phylogenetic Analyses ◽  
Glycosyl Hydrolase ◽  
Anaerobic Bacteria ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Lag Phase ◽  
Fermentation Products ◽  
Clostridium Clariflavum

ABSTRACTNine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to aClostridium clariflavumstrain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

scholarly journals Role of the bga1-Encoded Extracellular β-Galactosidase of Hypocrea jecorina in Cellulase Induction by Lactose

2005 ◽  
Vol 71 (2) ◽  
pp. 851-857 ◽  
Author(s):  
Bernhard Seiboth ◽  
Lukas Hartl ◽  
Noora Salovuori ◽  
Karin Lanthaler ◽  
Geoff D. Robson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Glycosyl Hydrolase ◽  
Cellulase Production ◽  
Lag Phase ◽  
Galactosidase Activity ◽  
Hypocrea Jecorina ◽  
Cellobiohydrolase I ◽  
Cellulase Induction

ABSTRACT Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major β-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular β-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Δbga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular β-galactosidase activity that was not encoded by bga1 but no intracellular β-galactosidase activity. In conclusion, cellulase production on lactose occurs when β-galactosidase activity levels are low but decreases as the β-galactosidase activities increase. The data indicate that bga1-encoded β-galactosidase activity is a critical factor for cellulase production on lactose.

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