Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120DC biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)-styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60-80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the main parameters influencing the biotransformation rate. Productivity was linearly dependent on the specific membrane area and on the tube wall thickness. On average volumetric productivities of 24 g L À1 aq day À1 with a maximum of 70 g L À1 aq day À1 and biomass concentrations of 45 g BDW L À1 aq have been achieved over long continuous process periods (!50 days) without reactor downtimes.

BackgroundOver the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli, Corynebacterium glutamicum, and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain.ResultsThe overexpression of kivd, coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps. sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 ± 1.5 mM isobutyric acid with a carbon yield of 0.12 ± 0.01 g gglc-1.ConclusionThe combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.

SummaryBiocatalytic processes often encounter problems due to toxic reactants and products, which reduce biocatalyst viability. Thus, robust organisms capable of tolerating or adapting towards such compounds are of high importance. This study systematically investigated the physiological response of Pseudomonas taiwanensis VLB120∆C biofilms when exposed to n‐butanol, one of the potential next generation biofuels as well as a toxic substance using microscopic and biochemical methods. Initially P. taiwanensis VLB120∆C biofilms did not show any observable growth in the presence of 3% butanol. Prolonged cultivation of 10 days led to biofilm adaptation, glucose and oxygen uptake doubled and consequently it was possible to quantify biomass. Complementing the medium with yeast extract and presumably reducing the metabolic burden caused by butanol exposure further increased the biomass yield. In course of cultivation cells reduced their size in the presence of n‐butanol which results in an enlarged surface‐to‐volume ratio and thus increased nutrient uptake. Finally, biofilm enhanced its extracellular polymeric substances (EPS) production when exposed to n‐butanol. The predominant response of these biofilms under n‐butanol stress are higher energy demand, increased biomass yield upon medium complements, larger surface‐to‐volume ratio and enhanced EPS production. Although we observed a distinct increase in biomass in the presence of 3% butanol it was not possible to cultivate P. taiwanensis VLB120∆C biofilms at higher n‐butanol concentrations. Thereby this study shows that biofilms are not per se tolerant against solvents, and need to adapt to toxic n‐butanol concentrations.

Thec hiral building block (S)-3-hydroxyisobutyric acid [(S)-3-HIBA] was produced either by conversion of isobutyric acid or directly from glucose utilizing cells of Pseudomonas taiwanensis VLB120 B83 T7 as catalysts.T his strain carriesapoint mutation in the gene encoding 3-hydroxyisobutyrate dehydrogenase (mmsB), leading to its inactivation.T he maximal specific activity in resting-cell biotransformations using isobutyric acid as substrate was 4.9 AE 0.4 Ug cdw À1 .O verexpression of the 2-ketoisovalerate pathwayg enes alsS,i lvC, and ilvD,a nd the introduction of kivd encoding 2-ketoacid decarboxylase resulted in the efficient fermentatives ynthesiso f( S )-3-HIBAd irectly from glucose.U pt o2 2mM( 2.3 g L À1 ) (S)-3-HIBAwere produceda t3 .7 AE 0.3 Ug cdw À1 in repeated batch experiments without observable product degradation. Utilizing ab iofilm reactor it was possible to continuously produce up to.O verall, the conversion of isobutyric acid to (S)-3-HIBAw as found to be the rate-limiting step,l eading to the accumulation of am ixture of (S)-3-hydroxyisobutyric acid andi sobutyric acid. This study demonstratesf or the first time the production of (S)-3-HIBAf rom renewable carbon in shake flasks and biofilm reactors and sets the stage for furthero ptimizations towards the efficient production of 3-HIBAa nd its derivatives in continuous fermentations.