Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7

Cadaverine is a C5 diamine monomer used for the production of bio-based polyamide 510. Cadaverine is produced by the decarboxylation of l-lysine using a lysine decarboxylase (LDC). In this study, we developed recombinant Escherichia coli strains for the expression of LDC from Hafnia alvei. The resulting recombinant XBHaLDC strain was used as a whole cell biocatalyst for the high-level bioconversion of l-lysine into cadaverine without the supplementation of isopropyl β-d-1-thiogalactopyranoside (IPTG) for the induction of protein expression and pyridoxal phosphate (PLP), a key cofactor for an LDC reaction. The comparison of results from enzyme characterization of E. coli and H. alvei LDC revealed that H. alvei LDC exhibited greater bioconversion ability than E. coli LDC due to higher levels of protein expression in all cellular fractions and a higher specific activity at 37 °C (1825 U/mg protein > 1003 U/mg protein). The recombinant XBHaLDC and XBEcLDC strains were constructed for the high-level production of cadaverine. Recombinant XBHaLDC produced a 1.3-fold higher titer of cadaverine (6.1 g/L) than the XBEcLDC strain (4.8 g/L) from 10 g/L of l-lysine. Furthermore, XBHaLDC, concentrated to an optical density (OD600) of 50, efficiently produced 136 g/L of cadaverine from 200 g/L of l-lysine (97% molar yield) via an IPTG- and PLP-free whole cell bioconversion reaction. Cadaverine synthesized via a whole cell biocatalyst reaction using XBHaLDC was purified to polymer grade, and purified cadaverine was successfully used for the synthesis of polyamide 510. In conclusion, an IPTG- and PLP-free whole cell bioconversion process of l-lysine into cadaverine, using recombinant XBHaLDC, was successfully utilized for the production of bio-based polyamide 510, which has physical and thermal properties similar to polyamide 510 synthesized from chemical-grade cadaverine.

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A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113480390 ◽

2013 ◽

Vol 18 (6) ◽

pp. 678-687 ◽

Cited By ~ 5

Author(s):

Hanna M. Dudek ◽

Petra Popken ◽

Edwin van Bloois ◽

Wouter A. Duetz ◽

Marco W. Fraaije

Keyword(s):

Detection Method ◽

Catalytic Properties ◽

Expression System ◽

Screening Method ◽

Whole Cell ◽

E Coli ◽

Coenzyme Regeneration ◽

Escherichia Coli Cells ◽

Phosphite Dehydrogenase ◽

Screening Site

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell–based screening method was validated by screening site–saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.

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Developing a dual-RCA microfluidic platform for sensitive E. coli O157:H7 whole-cell detections

Analytica Chimica Acta ◽

10.1016/j.aca.2020.06.046 ◽

2020 ◽

Vol 1127 ◽

pp. 79-88

Author(s):

Yuqian Jiang ◽

Zhenyu Qiu ◽

Tao Le ◽

Shan Zou ◽

Xudong Cao

Keyword(s):

Whole Cell ◽

Microfluidic Platform ◽

E Coli

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Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases

Molecules ◽

10.3390/molecules24142550 ◽

2019 ◽

Vol 24 (14) ◽

pp. 2550 ◽

Cited By ~ 4

Author(s):

Christoph Mähler ◽

Christian Burger ◽

Franziska Kratzl ◽

Dirk Weuster-Botz ◽

Kathrin Castiglione

Keyword(s):

Formation Rate ◽

Product Formation ◽

Regeneration System ◽

Initial Product ◽

Whole Cell ◽

Stirred Tank Reactors ◽

E Coli ◽

Space Time Yield ◽

Product Formation Rate ◽

Site Selective

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts’ activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.

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