Streamlined Pseudomonas taiwanensis VLB120 Chassis Strains with Improved Bioprocess Features

Abstract

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solventsensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis àla carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.

Benzoate synthesis from glucose or glycerol using engineered Pseudomonas taiwanensis

“…However, Pseudomonads were reported to tolerate higher amounts of catechol than produced during these experiments [19]. The concentrations produced by P. taiwanensis GRC3 Δ8ΔpykA -tap ΔcatBCA attTn7 ::P 14f -phdBCDE-4cl-pal should thus not yet lead to a high impairment of cellular fitness, especially considering that this strain is more solvent-tolerant [7]. Alternatively, the low titers of catechol may be due to its instability in the presence of oxygen and water [18,20].…”

“…Pseudomonas taiwanensis is a promising microbial cell factory, especially regarding the production of aromatics. This has been recently demonstrated by our group in multiple studies for de novosynthesis of phenol, 4-hydroxybenzoate, and trans -cinnamate [5][6][7][8]. In this study, a previously generated Pseudomonas taiwanensis trans -cinnamate overproducer was further engineered to enable benzoate production from renewable resources in a mineral medium without supplementation of complex substances or antibiotics.…”

Rational Engineering of Phenylalanine Accumulation in Pseudomonas taiwanensis to Enable High-Yield Production of Trans-Cinnamate

“…To further exploit the native potential of P. taiwanensis VLB120 as microbial cell factory, we recently engineered a genome-reduced variant of this strain which exhibited higher growth rates and enhanced biomass formation (Wynands et al, 2019). The benefit of genome reduction for potential aromatics production strains is underlined by the tolerance test in the presence of 30 mM t -cinnamate (Figure 1A).…”

“…In contrast to GRC1 and 3, GRC2 constitutively expresses ttgGHI genes that encode a solvent-efflux pump. Although this constitutive expression greatly increases the fitness of GRC2 in the presence of hydrophobic solvents such as toluene, it causes a fitness reduction in the absence of solvents (Wynands et al, 2019). This drawback is absent in the other two strains, where the whole ttg operon is absent (GRC1), or the regulatory genes ttgVW are included to induce the pump in response to solvents (GRC3).…”

“…To further exploit its potential as industrial production strain, we recently enhanced the bioprocess features of the solvent-tolerant strain Pseudomonas taiwanensis VLB120 by successive feature reduction (Wynands et al, 2019). The deletion of redundant genomic elements such as proviral segments, genes for biofilm formation and flagella expression and the megaplasmid pSTY resulted in strains with 15% enhanced growth rates and increased biomass yields, thereby improving the overall performance of the strain under bioprocess conditions.…”