Constitutively solvent-tolerant Pseudomonas taiwanensis VLB120∆ C∆ ttgV supports particularly high-styrene epoxidation activities when grown under glucose excess conditions.

Solvent-tolerant bacteria represent an interesting option to deal with the substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120Δ C. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120Δ C and VLB120Δ CΔ ttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance. Substrate inhibition kinetics was identified but was attenuated in two-liquid phase bioreactor setups. StyA fusion to the enhanced green fluorescent protein enabled precise enzyme level monitoring without affecting epoxidation activity. Glucose limitation compromised styAB expression and specific activities (30-40 U/g CDW for both strains), whereas unlimited batch cultivation enabled specific activities up to 180 U/g CDW for VLB120Δ CΔ ttgV strains, which is unrivaled for bioreactor-based whole-cell oxygenase biocatalysis. These extraordinarily high specific activities of constitutively solvent-tolerant P. taiwanensis VLB120∆ C∆ ttgV could be attributed to its high metabolic capacity, which also enabled high expression levels. This, together with the high product yields on glucose and biomass obtained qualifies the VLB120∆ ttgV strain as a highly attractive tool for the development of ecoefficient oxyfunctionalization processes and redox biocatalysis in general.

Insights into the Kinetics of the Resistance Formation of Bacteria against Ciprofloxacin Poly(2-methyl-2-oxazoline) Conjugates.

The influence on the resistance formation of polymers attached to antibiotics has rarely been investigated. In this study, ciprofloxacin (CIP) was conjugated to poly(2-methyl-2-oxazoline)s with an ethylene diamine end group (Me-PMOx28-EDA) via two different spacers (CIP modified with α,α'-dichloro- p-xylene-xCIP, CIP modified with chloroacetyl chloride-eCIP). The antibacterial activity of the conjugates against a number of bacterial strains shows a great dependence on the nature of the spacer. The Me-PMOx39-EDA-eCIP, containing a potentially cleavable linker, does not exhibit a molecular weight dependence on antibacterial activity in contrast to Me-PMOx27-EDA-xCIP. The resistance formation of both conjugates against Staphylococcus aureus and Escherichia coli was investigated. Both conjugates showed the potential to significantly delay the formation of resistant bacteria compared to the unmodified CIP. Closer inspection of a possible resistance mechanism by genome sequencing of the topoisomerase IV region of resistant S. aureus revealed that this bacterium mutates at the same position when building up resistance to CIP and to Me-PMOx27-EDA-xCIP. However, the S. aureus cells that became resistant against the polymer conjugate are fully susceptible to CIP. Thus, conjugation of CIP with PMOx seems to alter the resistance mechanism.

The application of constitutively solvent-tolerant P. taiwanensis VLB120ΔCΔttgV for stereospecific epoxidation of toxic styrene alleviates carrier solvent use.

For whole-cell biotransformations involving toxic organic compounds, two-liquid phase setups are typically applied employing an apolar extractive phase. Bis(2-ethylhexyl)phthalate (BEHP) has proven to be an ideal solvent for stereospecific styrene epoxidation with recombinant E. coli, providing excellent extractive properties and a high biocompatibility. In eco-efficiency evaluations, BEHP, however, has been identified as a critical factor regarding costs and environmental impact. In this study, the constitutive solvent tolerance of Pseudomonas taiwanensis VLB120ΔCΔttgV is shown to enable high specific activities (up to 180 U gCDW-1 ) and extensive reduction of the BEHP amount in two-liquid phase setups and thus to constitute an excellent tool to improve the environmental and economic efficiency of such processes. At a 90% reduction of carrier solvent use and accordingly increased aqueous styrene concentrations, this strain still showed reasonably high specific styrene epoxidation activities (100 U gCDW-1 ), while the solvent-adaptable wildtype strain immediately was toxified. A moderate 55% reduction of BEHP enabled a specific activity of 150 U gCDW-1 and thus represents a good trade-off between volumetric productivity maximization and environmental impact minimization. These results for the first time show a clear benefit of a solvent-tolerant compared to solvent-sensitive host strains and how such a benefit can be achieved.

Guiding bioprocess design by microbial ecology.

Industrial bioprocess development is driven by profitability and eco-efficiency. It profits from an early stage definition of process and biocatalyst design objectives. Microbial bioprocess environments can be considered as synthetic technical microbial ecosystems. Natural systems follow Darwinian evolution principles aiming at survival and reproduction. Technical systems objectives are eco-efficiency, productivity, and profitable production. Deciphering technical microbial ecology reveals differences and similarities of natural and technical systems objectives, which are discussed in this review in view of biocatalyst and process design and engineering strategies. Strategies for handling opposing objectives of natural and technical systems and for exploiting and engineering natural properties of microorganisms for technical systems are reviewed based on examples. This illustrates the relevance of considering microbial ecology for bioprocess design and the potential for exploitation by synthetic biology strategies.

Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.

The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

Whole-cell-based CYP153A6-catalyzed (S)-limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL.

Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)-limonene to (S)-perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8-PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate-limited in the two-liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g CDW-1). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single-phase and fivefold in two-liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)-perillyl alcohol L tot-1 within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co-expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates.