Measurement of strain‐dependent toxicity in the indene bioconversion using multiparameter flow cytometry

Abstract

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.

The efficiency of recombinantEscherichia coli as biocatalyst for stereospecific epoxidation

“…Interestingly, solvent tolerant P. putida S12 was even more sensitive to the membrane-destabilizing effect of styrene oxide. Such strain-dependent toxicities have also been reported for indene and its biooxidation products (Amanullah et al, 2003;Sikkema et al, 1995). The instability of P. putida S12 confirms the absolute need for preadaptation to achieve solvent tolerance, as demonstrated earlier for different solvent tolerant P. putida strains (Ramos-Gonzalez et al, 2001;Weber et al, 1993).…”

Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes

“…Bis-oxonol and PI Membrane potential and permeability [42] Organic acid stress Carboxyfluorescein diacetate (cFDA), PI, DiBAC 4 (3) and BODIPY FL vancomycin Membrane potential and permeability, esterase activity (metabolic activity) and cell-wall biosynthesis.…”

“…Stress responses analyzed by MPF include: substrate stress [42]; metabolite stress such as from organic acids [14 ,43] and solvents [44]; substrate starvation [45]; and temperature stress [46]. Survival of extracellular stress is often conferred by complex regulatory networks [15,33] that are not well understood, and upon selection for improvement, can resolve to be multigenic traits.…”

Asymmetric Dihydroxylation of Cinnamonitrile to trans‐3‐[(5S,6R)‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)

“…[31,39,40] At toxic substrate and product concentrations, the pores formed in the cytoplasmic membrane may be large enough to cause complete loss of the membrane integrity, resulting in cell death. [41] Formation of trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-acrylonitrile (1) started directly after the addition of cinnamonitrile with a rate of 0.29 U/g cdw until 6.5 h reaction time. Then net cell growth stopped, but the rate of product formation decreased only slowly until the end of the transformation period giving a final product concentration of 3.32 mM (0.55 g/L).…”