Pseudomonas taiwanensis biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors.

In this study, the biocatalytic performance of a Baeyer-Villiger monooxygenase (BVMO) catalyzing the reaction of cyclohexanone to ε-caprolactone was investigated in Pseudomonas biofilms. Biofilm growth and development of two Pseudomonas taiwanensis VLB120 variants, Ps_BVMO and Ps_BVMO_DGC, were evaluated in drip flow reactors (DFRs) and rotating bed reactors (RBRs). Engineering a hyperactive diguanylate cyclase (DGC) from Caulobacter crescentus into Ps_BVMO resulted in faster biofilm growth compared to the control Ps_BVMO strain in the DFRs. The maximum product formation rates of 92 and 87 g m-2 d-1 were observed for mature Ps_BVMO and Ps_ BVMO_DGC biofilms, respectively. The application of the engineered variants in the RBR was challenged by low biofilm surface coverage (50-60%) of rotating bed cassettes, side-products formation, oxygen limitation, and a severe drop in production rates with time. By implementing an active oxygen supply mode and a twin capillary spray feed, the biofilm surface coverage was maximized to 70-80%. BVMO activity was severely inhibited by cyclohexanol formation, resulting in a decrease in product formation rates. By controlling the cyclohexanone feed concentration at 4 mM, a stable product formation rate of 14 g m-2 d-1 and a substrate conversion of 60% was achieved in the RBR.

Mixed-trophies biofilm cultivation in capillary reactors.

The biocatalytic application of photoautotrophic organisms is a promising alternative for the production of biofuels and value-added compounds as they do not rely on carbohydrates as a source of carbon, electrons, and energy. Although the photoautotrophic organisms hold potential for the development of sustainable processes, suitable reactor concepts that allow high cell density (HCD) cultivation of photoautotrophic microorganisms are limited. Such reactors need a high surface to volume ratio to enhance light availability. Furthermore, the accumulation of high oxygen concentrations as a consequence of oxygenic photosynthesis, and its inhibitory effect on cell growth needs to be prevented. Here, we present a method for HCD cultivation of oxygenic phototrophs based on the co-cultivation of different trophies in a biofilm format to avoid high oxygen partial-pressure and attain HCDs of up to 51.8 gBDW L-1 on a lab scale. In this article, we show: •A robust method for mixed trophies biofilm cultivation in capillary reactors•Set-up and operation of a biofilm capillary reactor•A method to quantify oxygen in the continuous biofilm capillary reactor.

Data on mixed trophies biofilm for continuous cyclohexane oxidation to cyclohexanol using Synechocystis sp. PCC 6803.

Photosynthetic microorganisms offer promising perspectives for the sustainable production of value-added compounds. Nevertheless, the cultivation of phototrophic organisms to high cell densities (HCDs) is hampered by limited reactor concepts. Co-cultivation of the photoautotrophic Synechocystis sp. PCC 6803 and the chemoheterotrophic P. taiwanensis VLB 120 enabled HCDs up to 51.8 gCDW L-1. Respective biofilms have been grown as a biofilm in capillary flow-reactors, and oxygen evolution, total biomass, as well as the ratio of the two strains, have been followed under various cultivation conditions. Furthermore, biofilm formation on a microscopic level was analyzed via confocal laser scanning microscopy using a custom made flow-cell setup. The concept of mixed trophies co-cultivation was coupled to biotransformation, namely the oxyfunctionalization of cyclohexane to cyclohexanol. For benchmarking, the performance of the phototrophic reaction was compared to the chemical process, and to a biotechnological approach using a heterotrophic organism only. The data presented refer to our research paper "Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol" Hoschek et al., 2019.

Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803.

Oxygenase-containing cyanobacteria constitute promising whole-cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O2 , sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis-driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst-specific reaction rate is found to be light-dependent, reaching 26.3 ± 0.6 U gCDW -1 (U = µmol min-1 and cell dry weight [CDW]) at a light intensity of 150 µmolphotons m-2 s-1 . In situ substrate supply via a two-liquid phase system increases the initial specific activity to 39.2 ± 0.7 U gCDW -1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 gcyclohexanol gCDW -1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred-tank photobioreactor setup. In situ O2 generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration-independent light-driven oxyfunctionalization reactions involving highly toxic and volatile substrates.

Stabilization and scale-up of photosynthesis-driven ω-hydroxylation of nonanoic acid methyl ester by two-liquid phase whole-cell biocatalysis.

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O2 via photosynthetic water oxidation. Thus far, research on photosynthesis-driven bioprocesses mainly focuses on strain development and the proof of principle in small-scale biocatalytic reaction setups. This study investigates the long-term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω-hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity-independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two-liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7-fold increased initial specific activities (5.6 ± 0.1 U/gCDW ) and 7.6-fold increased specific yields on biomass (3.8 ± 0.1 mmolH-NAME /gCDW ) as compared with single aqueous phase biotransformations. Finally, the whole-cell biotransformation system was successfully scaled from glass tubes to a stirred-tank photobioreactor. This is the first study reporting the application of the two-liquid phase concept for efficient phototrophic whole-cell biocatalysis.

Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol.

Photosynthetic microorganisms have enormous potential to produce fuels and value-added compounds sustainably. Efficient cultivation concepts that enable optimal light and CO2 supply are necessary for the realization of high cell densities (HCDs), and subsequently for process implementation. We introduce capillary biofilm reactors with a high surface to volume ratio, and thus enhanced light availability, enabling HCDs of photo-autotrophic microorganisms. However, oxygenic photosynthesis leads to O2 accumulation in such systems, impairing biofilm growth. We combined O2 producing Synechocystis with O2 respiring Pseudomonas using proto-cooperation to achieve HCDs of up to 51.8 gBDW L-1. This concept was coupled to the challenging C-H oxyfunctionalization of cyclohexane to cyclohexanol with a remarkable conversion of >98% and selectivity of 100% (KA oil). High photoautotrophic biocatalyst concentrations were established and resulted in a productivity of 3.76 gcyclohexanol m-2 day-1, which was maintained for at least one month.

Overcoming the Gas-Liquid Mass Transfer of Oxygen by Coupling Photosynthetic Water Oxidation with Biocatalytic Oxyfunctionalization.

Gas-liquid mass transfer of gaseous reactants is a major limitation for high space-time yields, especially for O2 -dependent (bio)catalytic reactions in aqueous solutions. Herein, oxygenic photosynthesis was used for homogeneous O2 supply via in situ generation in the liquid phase to overcome this limitation. The phototrophic cyanobacterium Synechocystis sp. PCC6803 was engineered to synthesize the alkane monooxygenase AlkBGT from Pseudomonas putida GPo1. With light, but without external addition of O2 , the chemo- and regioselective hydroxylation of nonanoic acid methyl ester to ω-hydroxynonanoic acid methyl ester was driven by O2 generated through photosynthetic water oxidation. Photosynthesis also delivered the necessary reduction equivalents to regenerate the Fe2+ center in AlkB for oxygen transfer to the terminal methyl group. The in situ coupling of oxygenic photosynthesis to O2 -transferring enzymes now enables the design of fast hydrocarbon oxyfunctionalization reactions.

Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production.

The microbial production of isoprenoids has recently developed into a prime example for successful bottom-up synthetic biology or top-down systems biology strategies. Respective fermentation processes typically rely on growing recombinant microorganisms. However, the fermentative production of isoprenoids has to compete with cellular maintenance and growth for carbon and energy. Non-growing but metabolically active E. coli cells were evaluated in this study as alternative biocatalyst configurations to reduce energy and carbon loss towards biomass formation. The use of non-growing cells in an optimized fermentation medium resulted in more than fivefold increased specific limonene yields on cell dry weight and glucose, as compared to the traditional growing-cell-approach. Initially, the stability of the resting-cell activity was limited. This instability was overcome via the optimization of the minimal fermentation medium enabling high and stable limonene production rates for up to 8 h and a high specific yield of ≥50 mg limonene per gram cell dry weight. Omitting MgSO4 from the fermentation medium was very promising to prohibit growth and allow high productivities. Applying a MgSO4 -limitation also improved limonene formation by growing cells during non-exponential growth involving a reduced biomass yield on glucose and a fourfold increase in specific limonene yields on biomass as compared to non-limited cultures. The control of microbial growth via the medium composition was identified as a key but yet underrated strategy for efficient isoprenoid production. Biotechnol. Bioeng. 2016;113: 1305-1314. © 2015 Wiley Periodicals, Inc.

Coupling limonene formation and oxyfunctionalization by mixed-culture resting cell fermentation.

Metabolic engineering strategies mark a milestone for the fermentative production of bulk and fine chemicals. Yet, toxic products and volatile reaction intermediates with low solubilities remain challenging. Prominent examples are artificial multistep pathways like the production of perillyl acetate (POHAc) from glucose via limonene. For POHAc, these limitations can be overcome by mixed-culture fermentations. A limonene biosynthesis pathway and cytochrome P450 153A6 (CYP153A6) as regioselective hydroxylase are used in two distinct recombinant E. coli. POHAc formation from glucose in one recombinant cell was hindered by ineffective coupling of limonene synthesis and low rates of oxyfunctionalization. The optimization of P450 gene expression led to the formation of 6.20 ± 0.06 mg gcdw (-1) POHAc in a biphasic batch cultivation with glucose as sole carbon and energy source. Increasing the spatial proximity between limonene synthase and CYP153A6 by a genetic fusion of both enzymes changed the molar limonene/POHAc ratio from 3.2 to 1.6. Spatial separation of limonene biosynthesis from its oxyfunctionalization improved POHAc concentration 3.3-fold to 21.7 mg L(-1) as compared to a biphasic fermentation. Mixed-cultures of E. coli BL21 (DE3) containing the limonene biosynthesis pathway and E. coli MG1655 harboring either CYP153A6, or alternatively a cymene monooxygenase, showed POHAc formation rates of 0.06 or 0.11 U gcdw (-1) , respectively. This concept provides a novel framework for fermentative syntheses involving toxic, volatile, or barely soluble compounds or pathway intermediates.