Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Abstract

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 VLB120⌬C. 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 VLB120⌬C parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120⌬C⌬ttgV 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.

Catalytic Pseudomonas taiwanensis VLB120ΔC Biofilms Thrive in a Continuous Pure Styrene Generated by Multiphasic Segmented Flow in a Capillary Microreactor

“…The biofilm grown in three-phase aqueous-air-organic segmented flow exhibited enhanced tolerance compared to their suspended counterpart [14] and also displayed stable catalytic activities for longer period of time. Overall, this biofilm reactor is a promising format to perform catalysis involving toxic and poorly water soluble compounds.…”

“…Typically, organic compounds having logP o/w values (partition coefficient of a compound in an equimolar mixture of octanol and water) between 1 and 4 are considered to be toxic because they might interfere with the cellular membrane and disturb the physiological and functional integrity of the active cell [13]. While it was possible to grow biofilms in minimal medium under these conditions, the planktonic counterparts of Pseudomonas taiwanensis VLB120∆C needs complex nutrients such as yeast extracts or tryptone to support growth in the presence of a second phase of styrene 1% (v/v) [14]. The most favorable option to overcome substrate solubility and toxicity issues is to apply a second organic phase which acts as a substrate carrier phase to partition the substrate concentration below the toxicity limit and to deliver it continuously [7].…”

Microbial Production of Aliphatic ( S )-Epoxyalkanes by Using Rhodococcus sp. Strain ST-10 Styrene Monooxygenase Expressed in Organic-Solvent-Tolerant Kocuria rhizophila DC2201

“…In particular, Pseudomonas taiwanensis VLB120, from which SMOcoding genes were isolated, is well known as a solvent-tolerant microorganism. Volmer et al reported that the depletion of ttgV, which encodes a transcriptional repressor of solvent efflux pump gene expression, increased the solvent tolerance of P. taiwanensis VLB120 and biocatalytic activity of the epoxidation of styrene (37). This suggested that using organic solvent-tolerant microorganisms as heterologous expression hosts for RhSMO genes is a suitable strategy for improving our biocatalytic system for producing optically pure straight-chain (S)-epoxyalkanes.…”

Asymmetric bio-epoxidation catalyzed with the styrene monooxygenase from Pseudomonas sp. LQ26

“…Particularly, the SMO from Pseudomonas taiwanensis VLB120 (Volmer et al 2014) has been successfully scaled up in the production of enantiopure (S)-styrene oxide (Panke et al 2002). Further investigation Open Access *Correspondence: wuzhl@cib.ac.cn 1 Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, 9 South Renmin Road, 4th Section, Chengdu 610041, Sichuan, People's Republic of China Full list of author information is available at the end of the article has expanded its substrate spectrum to styrene derivatives and analogs (Lin et al 2011c;Liu et al 2015), as well as aliphatic olefins (Toda et al 2015).…”