Glycolate as alternative carbon source for Escherichia coli.

The physiology of different Escherichia coli stains was analyzed for growth with glycolate as a potentially promising sustainable sole source of carbon and energy. Different E. coli strains showed large differences regarding lag phases after provision of glycolate. Whereas E. coli W showed fast adaptation, E. coli BW25113, JM101, and BL21 (DE3) needed extensive time for adaption (up to 30 generations) until the attainable µmax was reached, which, at 30 °C, amounted to 0.20-0.25 h-1 for all strains. The overexpression of genes encoding glycolate degradation did neither overcome the need for adaptation of E. coli BL21 (DE3) nor improve growth of E. coli W. Rather, high level expression of proteins involved in uptake and initial degradation steps had an adverse effect on growth. Overall, the results show a promising capacity of E. coli strains for growth on glycolate.

Efficiency aspects of regioselective testosterone hydroxylation with highly active CYP450-based whole-cell biocatalysts.

Steroid hydroxylations belong to the industrially most relevant reactions catalysed by cytochrome P450 monooxygenases (CYP450s) due to the pharmacological relevance of hydroxylated derivatives. The implementation of respective bioprocesses at an industrial scale still suffers from several limitations commonly found in CYP450 catalysis, that is low turnover rates, enzyme instability, inhibition and toxicity related to the substrate(s) and/or product(s). Recently, we achieved a new level of steroid hydroxylation rates by introducing highly active testosterone-hydroxylating CYP450 BM3 variants together with the hydrophobic outer membrane protein AlkL into Escherichia coli-based whole-cell biocatalysts. However, the activity tended to decrease, which possibly impedes overall productivities and final product titres. In this study, a considerable instability was confirmed and subject to a systematic investigation regarding possible causes. In-depth evaluation of whole-cell biocatalyst kinetics and stability revealed a limitation in substrate availability due to poor testosterone solubility as well as inhibition by the main product 15ß-hydroxytestosterone. Instability of CYP450 BM3 variants was disclosed as another critical factor, which is of general significance for CYP450-based biocatalysis. Presented results reveal biocatalyst, reaction and process engineering strategies auguring well for industrial implementation of the developed steroid hydroxylation platform.

Enlighting Electron Routes In Oxyfunctionalizing Synechocystis sp. PCC 6803.

Phototrophic microorganisms, like cyanobacteria, are gaining attention as host organisms for biocatalytic processes with light as energy source and water as electron source. Redox enzymes, especially oxygenases, can profit from in-situ supply of co-substrates, i. e., reduction equivalents and O2 , by the photosynthetic light reaction. The electron transfer downstream of PS I to heterologous electron consuming enzymes in principle can involve NADPH, NADH, and/or ferredoxin, whereas most direct and efficient transfer is desirable. Here, we use the model organism Synechocystis sp. PCC 6803 to investigate, to what extent host and/or heterologous constituents are involved in electron transfer to a heterologous cytochrome P450 monooxygenase from Acidovorax sp. CHX100. Interestingly, in this highly active light-fueled cycloalkane hydroxylating biocatalyst, host-intrinsic enzymes were found capable of completely substituting the function of the Acidovorax ferredoxin reductase. To a certain extent (20 %), this also was true for the Acidovorax ferredoxin. These results indicate the presence of a versatile set of electron carriers in cyanobacteria, enabling efficient and direct coupling of electron consuming reactions to photosynthetic water oxidation. This will both simplify and promote the use of phototrophic microorganisms for sustainable production processes.

Light-driven redox biocatalysis on gram-scale in Synechocystis sp. PCC 6803 via an in vivo cascade.

The photosynthetic light reaction in cyanobacteria constitutes a highly attractive tool for productive biocatalysis, as it can provide redox reactions with high-energy reduction equivalents using sunlight and water as sources of energy and electrons, respectively. Here, we describe the first artificial light-driven redox cascade in Synechocystis sp. PCC 6803 to convert cyclohexanone to the polymer building block 6-hydroxyhexanoic acid (6-HA). Co-expression of a Baeyer-Villiger monooxygenase (BVMO) and a lactonase, both from Acidovorax sp. CHX100, enabled this two-step conversion with an activity of up to 63.1 ± 1.0 U/gCDW without accumulating inhibitory ε-caprolactone. Thereby, one of the key limitations of biocatalytic reactions, that is, reactant inhibition or toxicity, was overcome. In 2 L stirred-tank-photobioreactors, the process could be stabilized for 48 h, forming 23.50 ± 0.84 mm (3.11 ± 0.12 g/L) 6-HA. The high specificity enabling a product yield (YP/S ) of 0.96 ± 0.01 mol/mol and the remarkable biocatalyst-related yield of 3.71 ± 0.21 g6-HA /gCDW illustrate the potential of producing this non-toxic product in a synthetic cascade. The fine-tuning of the energy burden on the catalyst was found to be crucial, which indicates a limitation by the metabolic capacity of the cells possibly being compromised by biocatalysis-related reductant withdrawal. Intriguingly, energy balancing revealed that the biotransformation could tap surplus electrons derived from the photosynthetic light reaction and thereby relieve photosynthetic sink limitation. This study shows the feasibility of light-driven biocatalytic cascade operation in cyanobacteria and highlights respective metabolic limitations and engineering targets to unleash the full potential of photosynthesis.

Photosynthesis driven continuous hydrogen production by diazotrophic cyanobacteria in high cell density capillary photobiofilm reactors.

Hydrogen (H2) is a promising fuel in the context of climate neutral energy carriers and photosynthesis-driven H2-production is an interesting option relying mainly on sunlight and water as resources. However, this approach depends on suitable biocatalysts and innovative photobioreactor designs to maximize cell performance and H2 titers. Cyanobacteria were used as biocatalysts in capillary biofilm photobioreactors (CBRs). We show that biofilm formation/stability depend on light and CO2 availabilityH2 production rates correlate with these parameters but differ between Anabaena and Nostoc. We demonstrate that high light and corresponding O2 levels influence biofilm stability in CBR. By adjusting these parameters, biofilm formation/stability could be enhanced, and H2 formation was stable for weeks. Final biocatalyst titers reached up to 100 g l-1 for N. punctiforme atcc 29133 NHM5 and Anabaena sp. pcc 7120 AMC 414. H2 production rates were up to 300 µmol H2 l-1h-1 and 3 µmol H2 gcdw-1h-1 in biofilms.

Cyanobacteria as whole-cell factories: current status and future prospectives.

Cyanobacteria as phototrophic microorganisms bear great potential to produce chemicals from sustainable resources such as light and CO2. Most studies focus on either strain engineering or tackling metabolic constraints. Recently gained knowledge on internal electron and carbon fluxes and their regulation provides new opportunities to efficiently channel cellular resources toward product formation. Concomitantly, novel photobioreactor concepts are developed to ensure sufficient light supply. This review summarizes the newest developments in the field of cyanobacterial engineering to finally establish photosynthesis-based production processes. A holistic approach tackling genetic, metabolic, and biochemical engineering in parallel is considered essential to turn their application into an ecoefficient and economically feasible option for a future green bioeconomy.

Exploitation of Hetero- and Phototrophic Metabolic Modules for Redox-Intensive Whole-Cell Biocatalysis.

The successful realization of a sustainable manufacturing bioprocess and the maximization of its production potential and capacity are the main concerns of a bioprocess engineer. A main step towards this endeavor is the development of an efficient biocatalyst. Isolated enzyme(s), microbial cells, or (immobilized) formulations thereof can serve as biocatalysts. Living cells feature, beside active enzymes, metabolic modules that can be exploited to support energy-dependent and multi-step enzyme-catalyzed reactions. Metabolism can sustainably supply necessary cofactors or cosubstrates at the expense of readily available and cheap resources, rendering external addition of costly cosubstrates unnecessary. However, for the development of an efficient whole-cell biocatalyst, in depth comprehension of metabolic modules and their interconnection with cell growth, maintenance, and product formation is indispensable. In order to maximize the flux through biosynthetic reactions and pathways to an industrially relevant product and respective key performance indices (i.e., titer, yield, and productivity), existing metabolic modules can be redesigned and/or novel artificial ones established. This review focuses on whole-cell bioconversions that are coupled to heterotrophic or phototrophic metabolism and discusses metabolic engineering efforts aiming at 1) increasing regeneration and supply of redox equivalents, such as NAD(P/H), 2) blocking competing fluxes, and 3) increasing the availability of metabolites serving as (co)substrates of desired biosynthetic routes.

Heterologous Lactate Synthesis in Synechocystis sp. Strain PCC 6803 Causes a Growth Condition-Dependent Carbon Sink Effect.

Cyanobacteria are considered promising hosts for product synthesis directly from CO2 via photosynthetic carbon assimilation. The introduction of heterologous carbon sinks in terms of product synthesis has been reported to induce the so-called "carbon sink effect," described as the release of unused photosynthetic capacity by the introduction of additional carbon. This effect is thought to arise from a limitation of carbon metabolism that represents a bottleneck in carbon and electron flow, thus enforcing a downregulation of photosynthetic efficiency. It is not known so far how the cellular source/sink balance under different growth conditions influences the extent of the carbon sink effect and in turn product formation from CO2, constituting a heterologous carbon sink. We compared the Synechocystis sp. strain PCC 6803 wild type (WT) with an engineered lactate-producing strain (SAA023) in defined metabolic states. Unexpectedly, high-light conditions combined with carbon limitation enabled additional carbon assimilation for lactate production without affecting biomass formation. Thus, a strong carbon sink effect only was observed under carbon and thus sink limitation, but not under high-sink conditions. We show that the carbon sink effect was accompanied by an increased rate of alternative electron flow (AEF). Thus, AEF plays a crucial role in the equilibration of source/sink imbalances, presumably via ATP/NADPH balancing. This study emphasizes that the evaluation of the biotechnological potential of cyanobacteria profits from cultivation approaches enabling the establishment of defined metabolic states and respective quantitative analytics. Factors stimulating photosynthesis and carbon fixation are discussed. IMPORTANCE Previous studies reported various and differing effects of the heterologous production of carbon-based molecules on photosynthetic and growth efficiency of cyanobacteria. The typically applied cultivation in batch mode, with continuously changing growth conditions, however, precludes a clear differentiation between the impact of cultivation conditions on cell physiology and effects related to the specific nature of the product and its synthesis pathway. In this study, we employed a continuous cultivation system to maintain defined source/sink conditions and thus metabolic states. This allowed a systematic and quantitative analysis of the effect of NADPH-consuming lactate production on photosynthetic and growth efficiency. This approach enables a realistic evaluation of the biotechnological potential of engineered cyanobacterial strains. For example, the quantum requirement for carbon production was found to constitute an excellent indicator of the source/sink balance and thus a key parameter for photobioprocess optimization. Such knowledge is fundamental for rational and efficient strain and process development.

Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria.

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer-Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.

Rational orthologous pathway and biochemical process engineering for adipic acid production using Pseudomonas taiwanensis VLB120.

Microbial bioprocessing based on orthologous pathways constitutes a promising approach to replace traditional greenhouse gas- and energy-intensive production processes, e.g., for adipic acid (AA). We report the construction of a Pseudomonas taiwanensis strain able to efficiently convert cyclohexane to AA. For this purpose, a recently developed 6-hydroxyhexanoic acid (6HA) synthesis pathway was amended with alcohol and aldehyde dehydrogenases, for which different expression systems were tested. Thereby, genes originating from Acidovorax sp. CHX100 and the XylS/Pm regulatory system proved most efficient for the conversion of 6HA to AA as well as the overall cascade enabling an AA formation activity of up to 48.6 ± 0.2 U gCDW-1. The optimization of biotransformation conditions enabled 96% conversion of 10 mM cyclohexane with 100% AA yield. During recombinant gene expression, the avoidance of glucose limitation was found to be crucial to enable stable AA formation. The biotransformation was then scaled from shaking flask to a 1 L bioreactor scale, at which a maximal activity of 22.6 ± 0.2 U gCDW-1 and an AA titer of 10.2 g L-1 were achieved. The principal feasibility of product isolation was shown by the purification of 3.4 g AA to a purity of 96.1%. This study presents the efficient bioconversion of cyclohexane to AA by means of a single strain and thereby sets the basis for an environmentally benign production of AA and related polymers such as nylon 6,6.

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase.

Molecular hydrogen (H2) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H2 production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H2 syntheses rely mainly on light, water, and CO2 and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H2-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O2) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O2-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O2-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O2 sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O2 tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH+ to utilize H2 as sole electron source to fix CO2. Syn_ReSH+ also was able to produce H2 under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H2 production.

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3-fold higher) and kcat (4.8-fold lower) compared with corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3-fold higher). The unfavorable KS and kcat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher KS , but 1.3- and 4.2-fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

One-pot synthesis of 6-aminohexanoic acid from cyclohexane using mixed-species cultures.

6-Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA-approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis. A mixed-species approach turned out to be most promising. Thereby, Pseudomonas taiwanensis VLB120 strains harbouring an upstream cascade converting cyclohexane to either є-caprolactone (є-CL) or 6-hydroxyhexanoic acid (6HA) were combined with Escherichia coli JM101 strains containing the corresponding downstream cascade for the further conversion to 6AHA. ε-CL was found to be a better 'shuttle molecule' than 6HA enabling higher 6AHA formation rates and yields. Mixed-species reaction performance with 4 g l-1 biomass, 10 mM cyclohexane, and an air-to-aqueous phase ratio of 23 combined with a repetitive oxygen feeding strategy led to complete substrate conversion with 86% 6AHA yield and an initial specific 6AHA formation rate of 7.7 ± 0.1 U gCDW -1 . The same cascade enabled 49% 7-aminoheptanoic acid yield from cycloheptane. This combination of rationally engineered strains allowed direct 6AHA production from cyclohexane in one pot with high conversion and yield under environmentally benign conditions.

Rational Engineering of a Multi-Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis.

The current industrial production of polymer building blocks such as ε-caprolactone (ε-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco-efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions. Based on kinetic and thermodynamic analyses of the individual enzymatic steps, a 4-step enzymatic cascade in Pseudomonas taiwanensis VLB120 is rationally engineered via stepwise biocatalyst improvement on the genetic level. It is found that the intermediate product cyclohexanol severely inhibits the cascade which could be optimized by enhancing the expression level of downstream enzymes. The integration of a lactonase enables exclusive 6HA formation without side products. The resulting biocatalyst shows a high activity of 44.8 ± 0.2 U gCDW -1 and fully converts 5 mm cyclohexane to 6HA within 3 h. This platform organism can now serve as a basis for the development of greener production processes for polycaprolactone and related polymers.

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120.

Cytochrome P450 monooxygenases (Cyps) effectively catalyze the regiospecific oxyfunctionalization of inert C-H bonds under mild conditions. Due to their cofactor dependency and instability in isolated form, oxygenases are preferably applied in living microbial cells with Pseudomonas strains constituting potent host organisms for Cyps. This study presents a holistic genetic engineering approach, considering gene dosage, transcriptional, and translational levels, to engineer an effective Cyp-based whole-cell biocatalyst, building on recombinant Pseudomonas taiwanensis VLB120 for cyclohexane hydroxylation. A lac-based regulation system turned out to be favorable in terms of orthogonality to the host regulatory network and enabled a remarkable specific whole-cell activity of 34 U gCDW -1. The evaluation of different ribosomal binding sites (RBSs) revealed that a moderate translation rate was favorable in terms of the specific activity. An increase in gene dosage did only slightly elevate the hydroxylation activity, but severely impaired growth and resulted in a large fraction of inactive Cyp. Finally, the introduction of a terminator reduced leakiness. The optimized strain P. taiwanensis VLB120 pSEVA_Cyp allowed for a hydroxylation activity of 55 U gCDW -1. Applying 5 mM cyclohexane, molar conversion and biomass-specific yields of 82.5% and 2.46 mmolcyclohexanol gbiomass -1 were achieved, respectively. The strain now serves as a platform to design in vivo cascades and bioprocesses for the production of polymer building blocks such as ε-caprolactone.

Regulatory systems for gene expression control in cyanobacteria.

As photosynthetic microbes, cyanobacteria are attractive hosts for the production of high-value molecules from CO2 and light. Strategies for genetic engineering and tightly controlled gene expression are essential for the biotechnological application of these organisms. Numerous heterologous or native promoter systems were used for constitutive and inducible expression, yet many of them suffer either from leakiness or from a low expression output. Anyway, in recent years, existing systems have been improved and new promoters have been discovered or engineered for cyanobacteria. Moreover, alternative tools and strategies for expression control such as riboswitches, riboregulators or genetic circuits have been developed. In this mini-review, we provide a broad overview on the different tools and approaches for the regulation of gene expression in cyanobacteria and explain their advantages and disadvantages.

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.

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.

Anaerobic C-H Oxyfunctionalization: Coupling of Nitrate Reduction and Quinoline Hydroxylation in Recombinant Pseudomonas putida.

Whole-cell biocatalysis for C-H oxyfunctionalization depends on and is often limited by O2 mass transfer. In contrast to oxygenases, molybdenum hydroxylases use water instead of O2 as an oxygen donor and thus have the potential to relieve O2 mass transfer limitations. Molybdenum hydroxylases may even allow anaerobic oxyfunctionalization when coupled to anaerobic respiration. To evaluate this option, the coupling of quinoline hydroxylation to denitrification is tested under anaerobic conditions employing Pseudomonas putida (P. putida) 86, capable of aerobic growth on quinoline. P. putida 86 reduces both nitrate and nitrite, but at low rates, which does not enable significant growth and quinoline hydroxylation. Introduction of the nitrate reductase from Pseudomonas aeruginosa enables considerable specific quinoline hydroxylation activity (6.9 U gCDW -1 ) under anaerobic conditions with nitrate as an electron acceptor and 2-hydroxyquinoline as the sole product (further metabolization depends on O2 ). Hydroxylation-derived electrons are efficiently directed to nitrate, accounting for 38% of the respiratory activity. This study shows that molybdenum hydroxylase-based whole-cell biocatalysts enable completely anaerobic carbon oxyfunctionalization when coupled to alternative respiration schemes such as nitrate respiration.