scholarly journals Repurposing chloramphenicol acetyltransferase for a robust and efficient designer ester biosynthesis platform

2020 ◽  
Author(s):  
Hyeongmin Seo ◽  
Jong-Won Lee ◽  
Richard J. Giannone ◽  
Noah J. Dunlap ◽  
Cong T. Trinh
Keyword(s):  
Elevated Temperatures ◽  
Isoamyl Acetate ◽  
Thermophilic Bacterium ◽  
Cellulosic Biomass ◽  
E Coli ◽  
Aromatic Esters ◽  
Fed Batch Fermentation ◽  
Mesophilic Bacterium ◽  
Ester Biosynthesis ◽  
Microbial Biosynthesis

AbstractRobust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as an interchangeable metabolic module for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and repurposed chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacterium Escherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinant E. coli was also capable of simulating the ester profile of roses with high conversion (> 97%) and titer (> 1 g/L) from fermentable sugars at 37°C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacterium Clostridium thermocellum harboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50°C) due to the engineered enzyme’s remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.

Characterization of Thermophilic Bacteria Using Surface-Enhanced Raman Scattering

2008 ◽  
Vol 62 (11) ◽  
pp. 1226-1232 ◽  
Author(s):  
Mustafa Çulha ◽  
Ahmet Adigüzel ◽  
M. MÜGE Yazici ◽  
Mehmet Kahraman ◽  
Fikrettin Slahin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Raman Scattering ◽  
Elevated Temperatures ◽  
Thermophilic Bacteria ◽  
Surface Enhanced Raman Scattering ◽  
Molecular Structures ◽  
E Coli ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Enhanced Raman Scattering

Surface-enhanced Raman scattering (SERS) can provide molecular-level information about the molecules and molecular structures in the vicinity of nanostructured noble metal surfaces such as gold and silver. The three thermophilic bacteria Bacillus licheniformis, Geobacillus stearothermophilus, and Geobacillus pallidus, a Gram-negative bacterium E. coli, and a Gram-positive bacterium B. megaterium are comparatively characterized using SERS. The SERS spectra of thermophilic bacteria are similar, while they show significant differences compared to E. coli and B. megaterium. The findings indicate that a higher number of thiol residues and possible S–S bridges are present in the cell wall structure of thermophilic bacteria, providing their stability at elevated temperatures. Incubating the thermophilic bacteria with colloidal silver suspension at longer times improved the bacteria–silver nanoparticle interaction kinetics, while increased temperature does not have a pronounced effect on spectral features. A tentative assignment of the SERS bands was attempted for thermophilic bacteria. The results indicate that SERS can be a useful tool to study bacterial cell wall molecular differences.

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Elevated Temperatures ◽  
Bacterial Genome ◽  
Thermophilic Bacterium ◽  
Geobacillus Thermodenitrificans ◽  
Temperature Range ◽  
Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Genomic and phenotypic analysis of heat and sanitizer resistance in Escherichia coli from beef in relation to the locus of heat resistance

2021 ◽  
Author(s):  
Xianqin Yang ◽  
Frances Tran ◽  
Peipei Zhang ◽  
Hui Wang
Keyword(s):  
Escherichia Coli ◽  
Heat Resistance ◽  
Phylogenetic Relationships ◽  
Core Genome ◽  
Elevated Temperatures ◽  
Resistant Bacteria ◽  
Phenotypic Analysis ◽  
Decimal Reduction Time ◽  
E Coli ◽  
Clade 1

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships, and genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n=24) were whole genome-sequenced by short- and long-reads. LHR-negative isolates (n=18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C ( D 60°C ) and susceptibility to E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and negative isolates were well separated on the core genome tree, with 22/24 of the positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D 60°C values, were clonal, as determined by subtyping (MLST, core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (p<0.001) median genome size by 0.3 Mbp (5.0 vs 4.7 Mbp), and overrepresentation of genes in plasmid maintenance, stress response and cryptic prophages, but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71±0.02 and 0.65±0.02 logOD h −1 for LHR-positive and negative isolates. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and negative isolates. Importance Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli , and that strains with certain genetic background are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes in virulence and epithelial attachment. The phylogenetic relationships among the isolates point towards multiple mechanisms for acquiring LHR, likely prior to their deposition on meat.

Fate of Escherichia coli O157:H7 in the Presence of Indigenous Microorganisms on Commercially Packaged Baby Spinach, as Impacted by Storage Temperature and Time†

2009 ◽  
Vol 72 (10) ◽  
pp. 2038-2045 ◽  
Author(s):  
YAGUANG LUO ◽  
QIANG HE ◽  
JAMES L. McEVOY ◽  
WILLIAM S. CONWAY
Keyword(s):  
Escherichia Coli ◽  
Product Quality ◽  
Elevated Temperatures ◽  
Storage Temperature ◽  
Escherichia Coli O157 ◽  
Indigenous Microorganisms ◽  
E Coli ◽  
Psychrotrophic Bacteria ◽  
Survival And Growth

This study investigated the effect of storage temperature and time on the survival and growth of Escherichia coli O157:H7, the growth of indigenous microorganisms, and the changes in product quality of packaged baby spinach. Commercial packages of spinach within 2 days of processing were cut open at one end, sprayed with fine mists of E. coli O157:H7 inoculum, resealed, and then stored at 1, 5, 8, and 12°C for 12 days until their labeled best-if-used-by dates. Microbial enumeration and product quality evaluation were conducted on day(s) 0, 3, 6, 9, and 12 postinoculation. Spinach held at 12°C supported significant (P &lt; 0.001) E. coli O157:H7 growth, with a 1.0-log CFU/g increase within 3 days postinoculation, which was followed by additional growth during continued storage. E. coli O157:H7 grew slowly when held at 8°C, with a significant (P &lt; 0.01) level of growth reached after 6 days of storage. However, on products held at 1 and 5°C, E. coli O157:H7 populations declined significantly (P &lt; 0.01 and P &lt; 0.001, respectively) within 3 days of storage. Aerobic mesophilic bacteria, psychrotrophic bacteria, and yeast and mold populations increased significantly at all storage temperatures, with more growth on products held at elevated temperatures. Product quality scores remained high within the first 6 days of storage, with a sharp decline noted on samples held at 12°C on day 9. Results suggest that E. coli O157:H7 can grow significantly on commercially packaged spinach held at 8°C or above before significant product quality deterioration occurs.

scholarly journals Differential expression of small RNAs under chemical stress and fed-batch fermentation in E. coli

BMC Genomics ◽  
2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Martin Holm Rau ◽  
Klara Bojanovič ◽  
Alex Toftgaard Nielsen ◽  
Katherine S. Long
Keyword(s):  
Differential Expression ◽  
Small Rnas ◽  
Batch Fermentation ◽  
Chemical Stress ◽  
Fed Batch ◽  
E Coli ◽  
Fed Batch Fermentation

Co-Expression of Threonine Dehydratase and Acetolactate Synthase in Escherichia Coli for L-Isoleucine Production

2014 ◽  
Vol 989-994 ◽  
pp. 997-1002 ◽  
Author(s):  
Jian Wang ◽  
Jia Kai Sun ◽  
Qing Yang Xu
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Carbon Flux ◽  
Batch Fermentation ◽  
Acetolactate Synthase ◽  
Fed Batch ◽  
Threonine Dehydratase ◽  
E Coli ◽  
Fed Batch Fermentation ◽  
Fermentation Period

Metabolic engineering ofCorynebacterium glutamicumhas sought to divert carbon into L-isoleucine. However, the fermentation period of this strain is long. TheC.glutamicumYILW strain (LeuL, AHVr, SGr, Leu-MEr) was previously derived by repeated compound mutagenesis which could accumulate 20.2 g/L L-isoleucine in a 5-L jar fermentor. Overexpression of the threonine dehydratase gene (ilvA) fromCorynebacterium glutamicumYILW and coexpression of threonine dehydratase and acetolactate synthase (ilvBN) from it were employed to divert carbon flux toward L-isoleucine. The strainE. coliTRFC with the expression ofilvA could accumulate L-isoleucine of 6.8 g/L without accumulation of any L-threonine by fed-batch fermentation in a 5-L jar fermentor. However, the production of L-isoleucine by the strainE.coliTRFC with the co-expression ofilvA andilvBN was decreased by 19.1%, and the production of L-valine was increased by 40% compared with that ofE. coliTRFC with the expression ofilvA.

scholarly journals Engineering Deinococcus geothermalis for Bioremediation of High-Temperature Radioactive Waste Environments

2003 ◽  
Vol 69 (8) ◽  
pp. 4575-4582 ◽  
Author(s):  
Hassan Brim ◽  
Amudhan Venkateswaran ◽  
Heather M. Kostandarithes ◽  
James K. Fredrickson ◽  
Michael J. Daly
Keyword(s):  
High Temperature ◽  
Radioactive Waste ◽  
Elevated Temperatures ◽  
Deinococcus Radiodurans ◽  
Nitrilotriacetic Acid ◽  
Thermophilic Bacterium ◽  
Prospective Development ◽  
Deinococcus Geothermalis ◽  
Radiation Resistant

ABSTRACT Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.

Selection of In Vivo Expressed Genes of Escherichia coli O157:H7 Strain EDL933 in Ground Meat under Elevated Temperature Conditions

2012 ◽  
Vol 75 (10) ◽  
pp. 1743-1750 ◽  
Author(s):  
ANDREA KROJ ◽  
HERBERT SCHMIDT
Keyword(s):  
Escherichia Coli ◽  
Elevated Temperatures ◽  
Genomic Library ◽  
Transport Processes ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
E Coli ◽  
Selection Of

Enterohemorrhagic Escherichia coli O157:H7 strains are important foodborne pathogens that are often transmitted to humans by the ingestion of raw or undercooked meat of bovine origin. To investigate adaptation of this pathogen during persistence and growth in ground meat, we established an in vivo expression technology model to identify genes that are expressed during growth in this food matrix under elevated temperatures (42°C). To improve on the antibiotic-based selection method, we constructed the promoter trap vector pAK-1, containing a promoterless kanamycin resistance gene. A genomic library of E. coli O157:H7 strain EDL933 was constructed in pAK-1 and used for promoter selection in ground meat. The 20 in vivo expressed genes identified were associated with transport processes, metabolism, macromolecule synthesis, and stress response. For most of the identified genes, only hypothetical functions could be assigned. The results of our study provide the first insights into the complex response of E. coli O157:H7 to a ground meat environment under elevated temperatures and establish a suitable vector for promoter studies or selection of in vivo induced promoters in foods such as ground meat.

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