Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds

Abstract

A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli, through the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase-mediated C-C bond formation, has been investigated. An enzymatic toolbox consisting of alcohol dehydrogenase AlkJ from Pseudomonas putida and the dihydroxyacetone-/hydroxyacetone-accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed at the genetic and process levels. Three different arrangements of the alkJ and fsa1-A129S genes in operon, monocistronic, and pseudo-operon configuration were tested. The last of these proved to be most beneficial with regard to bacterial growth and protein expression levels. The optimized whole-cell catalyst, combined with a refined solid-phase extraction downstream purification protocol, provides diastereomerically pure carbohydrate derivatives that can be isolated in up to 91 % yield over two reaction steps.

LuxAB-Based Microbial Cell Factories for the Sensing, Manufacturing and Transformation of Industrial Aldehydes

“…For the construction of the biosensor device, the luxAB coding region was amplified from pAK400c/iluxAB_Cm r , which was previously constructed for the integration of luxAB into the A. baylyi genome and the monitoring of intracellular aldehyde levels (≤C 18 ) [2,48]. Subcloning of luxAB under the control of a constitutive T5 promoter into the target pCDFDuet-1 vector was facilitated by a modified sequence and ligation-independent cloning (SLIC) procedure [49,50], yielding pLuxAB (Table S10). Functionality of the biosensor system was tested in E. coli RARE resting cells (RCs) as described in this study.…”

“…In this study, plasmids were assembled from two linear DNA fragments via homologous overhangs attached by PCR, following adapted protocols from Li et al [76] or Wiesinger et al [50]. The latter employed a seamless and ligation-independent cloning extract (SLiCE) that was prepared according to Zhang et al from E. coli TOP10 cultures [49].…”

“…Protein expression was confirmed by 12.5% (ω/ν) SDS-PAGE analysis of whole-cell samples normalized to OD 600 = 7.0, using the Mini-PROTEAN electrophoresis system (Bio-Rad, Feldkirchen, Germany) and following standard protocols (e.g., [50]). Gels were stained with InstantBlue TM Protein Stain (Expedeon, Heidelberg, Germany).…”

“…RCs co-expressing LuxAB and the enzyme of interest were prepared as described above. To 198 µL RCs (OD 600 ≈ 10.0) per well, 2 µL stock solution of the target substrate (0.1 M in dimethyl sulfoxide or ethanol) were added to a final concentration of 1 mM substrate (V total = 200 µL containing 1% (ν/ν) organic co-solvent) in 96-well plates (flat bottom, black polystyrene [50,65,76]; Greiner Bio-One, Frickenhausen, Germany). It was mixed gently and the bioluminescence measured immediately on a Varioskan TM LUX multimode plate reader (Thermo Fisher Scientific).…”

In situ Generation of Aldehydes for Subsequent Biocatalytic Cascade Reactions in Whole Cells

“…A crucial part of the realization of any enzymatic cascade is the evaluation of single enzyme activity. The oxidizing/reducing activity of the enzymes CAR Ni and AlkJ has been investigated in our previous studies [8b,11] . In order to expand the concept, new enzymes for the catalysis of three different reaction types were investigated.…”