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 15β-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.

The implementation of biocatalytic steroid hydroxylation processes plays a crucial role in the pharmaceutical industry due to a plethora of medicative effects of hydroxylated steroid derivatives and their crucial role in drug approval processes. Cytochrome P450 monooxygenases (CYP450s) typically constitute the key enzymes catalyzing these reactions, but commonly entail drawbacks such as poor catalytic rates and the dependency on additional redox proteins for electron transfer from NAD(P)H to the active site. Recently, these bottlenecks were overcome by equipping Escherichia coli cells with highly active variants of the self-sufficient single-component CYP450 BM3 together with hydrophobic outer membrane proteins facilitating cellular steroid uptake. The combination of the BM3 variant KSA14m and the outer membrane pore AlkL enabled exceptionally high testosterone hydroxylation rates of up to 45 U gCDW-1 for resting (i.e., living but non-growing) cells. However, a rapid loss of specific activity heavily compromised final product titers and overall space-time yields. In this study, several stabilization strategies were evaluated on enzyme-, cell-, and reaction level. However, neither changes in biocatalyst configuration nor variation of cultivation media, expression systems, or inducer concentrations led to considerable improvement. This qualified the so-far used genetic construct pETM11-ksa14m-alkL, M9 medium, and the resting-cell state as the best options enabling comparatively efficient activity along with fast growth prior to biotransformation. In summary, we report several approaches not enabling a stabilization of the high testosterone hydroxylation rates, providing vital guidance for researchers tackling similar CYP450 stability issues. A comparison with more stable natively steroid-hydroxylating CYP106A2 and CYP154C5 in equivalent setups further highlighted the high potential of the investigated CYP450 BM3-based whole-cell biocatalysts. The immense and continuously developing repertoire of enzyme engineering strategies provides promising options to stabilize the highly active biocatalysts.

Biocatalytic steroid hydroxylation processes are highly relevant for the pharmaceutical industry but are still limited by low specific activities and stabilities. For testosterone hydroxylation, high activities were achieved with recombinant Escherichia coli containing a highly active CYP450-BM3 variant and outer membrane porins. Enzyme instability and substrate mass transfer were identified as further limitations, for which biocatalyst and reaction engineering strategies are discussed.

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 mu (max) was reached, which, at 30 degrees 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.

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.

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 O-2, 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.

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 pa-rameters 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 mu mol H2 l -1h- 1 and 3 mu mol H2 gcdw-1 h- 1 in biofilms.

•Capillary photobiofilm reactors allow high density cultivation of cyanobacteria.•Continuous hydrogen production by diazotrophic cyanobacteria.•Environmental factors determine biofilm stability and hydrogen production.

SummaryThe 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.

Chemie Ingenieur TechnikVolume 94, Issue 9 p. 1260-1260 Poster Development of a photosynthetically driven biocatalyst for the conversion of cyclohexane to ε-caprolactone N. Siebert, Corresponding Author N. Siebert nina_antonia.siebert@uni-leipzig.de Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, GermanyCorrespondence: N. Siebert (nina_antonia.siebert@uni-leipzig.de), Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, GermanySearch for more papers by this authorA. Tüllinghoff, A. Tüllinghoff Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorB. Bühler, B. Bühler Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorR. Karande, R. Karande Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, Germany Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this author N. Siebert, Corresponding Author N. Siebert nina_antonia.siebert@uni-leipzig.de Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, GermanyCorrespondence: N. Siebert (nina_antonia.siebert@uni-leipzig.de), Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, GermanySearch for more papers by this authorA. Tüllinghoff, A. Tüllinghoff Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorB. Bühler, B. Bühler Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorR. Karande, R. Karande Leipzig University, Interfaculty Centre for Bioactive Matter, Johannisallee 21–23, 04103 Leipzig, Germany Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this author First published: 25 August 2022 https://doi.org/10.1002/cite.202255342AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume94, Issue9Special Issue: (Bio)Process Engineering – a Key to Sustainable Development: ProcessNet and DECHEMA-BioTechNet Jahrestagungen 2022 together with 13th ESBES SymposiumSeptember 2022Pages 1260-1260 RelatedInformation

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.

Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O 2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a P nrsB-(Ni 2+ )–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U g CDW −1 . Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mg BVMO −1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW −1 , a maximal volumetric productivity of 0.21 ± 0.1 gL −1 h −1 , and the formation of 1.3 ± 0.1 gL −1 of ε -caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.

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.

Chemie Ingenieur TechnikVolume 94, Issue 9 p. 1258-1258 Poster Glycolic acid as an alternative carbon and energy source for redox biocatalysis S. Höhmann, S. Höhmann Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorN. Ihle, N. Ihle Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorA. Schmid, A. Schmid Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorB. Bühler, Corresponding Author B. Bühler bruno.buehler@ufz.de Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanyCorrespondence: B. Bühler (bruno.buehler@ufz.de), Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this author S. Höhmann, S. Höhmann Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorN. Ihle, N. Ihle Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorA. Schmid, A. Schmid Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this authorB. Bühler, Corresponding Author B. Bühler bruno.buehler@ufz.de Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanyCorrespondence: B. Bühler (bruno.buehler@ufz.de), Helmholtz Centre for Environmental Research, Solar Materials, Permoserstr. 15, 04318 Leipzig, GermanySearch for more papers by this author First published: 25 August 2022 https://doi.org/10.1002/cite.202255380AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume94, Issue9Special Issue: (Bio)Process Engineering – a Key to Sustainable Development: ProcessNet and DECHEMA-BioTechNet Jahrestagungen 2022 together with 13th ESBES SymposiumSeptember 2022Pages 1258-1258 RelatedInformation

Cyanobacteria have raised great interest in biotechnology due to theirpotential for a sustainable, photosynthesis-driven production of fuels and value-addedchemicals. This has led to a concomitant development of molecular tools to engineerthe metabolism of those organisms. In this regard, however, even cyanobacterial modelstrains lag behind compared to their heterotrophic counterparts. For instance,replicative shuttle vectors that allow gene transfer independent of recombination intohost DNA are still scarce. Here, we introduce the pSOMA shuttle vector seriescomprising 10 synthetic plasmids for comprehensive genetic engineering ofSynechocystissp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 andpCB2.4, each combined with a replicon fromEscherichia coli,different selection markersas well as features facilitating molecular cloning and the insulated introduction of geneexpression cassettes. We made use of genes encoding greenfluorescent protein (GFP) and a Baeyer-Villiger monooxygenase(BVMO) to demonstrate functional gene expression from the pSOMA plasmidsin vivo. Moreover, we demonstrate the expression ofdistinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between thetwo pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into thefilamentous model strainAnabaenasp. PCC 7120 is generally possible, which is encouraging to further explore the range ofcyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays anattractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups andto perform extensive metabolic engineering of cyanobacteria

The implementation of biocatalytic steroid hydroxylation processes at an industrial scale still suffers from low conversion rates. In this study, we selected variants of the self-sufficient cytochrome P450 monooxygenase BM3 from Bacillus megaterium (BM3) for the hydroxylation of testosterone either at the 2β- or 15β-position. Recombinant Escherichia coli cells were used as biocatalysts to provide a protective environment for recombinant enzymes and to ensure continuous cofactor recycling via glucose catabolism. However, only low initial whole-cell testosterone conversion rates were observed for resting cells. Results obtained with different biocatalyst formats (permeabilized cells, cell-free extracts, whole cells) indicated a limitation in substrate uptake, most likely due to the hydrophilic character of the outer membrane of E. coli. Thus, we co-expressed nine genes encoding hydrophobic outer membrane proteins potentially facilitating steroid uptake. Indeed, the application of four candidates led to increased initial testosterone hydroxylation rates. Respective whole-cell biocatalysts even exceeded activities obtained with permeabilized cells or cell-free extracts. The highest activity of 34 U gCDW −1 was obtained for a strain containing the hydrophobic outer membrane protein AlkL from Pseudomonas putida GPo1 and the BM3 variant KSA14m. Overall, we show that the straightforward application of hydrophobic outer membrane pores can boost whole-cell steroid conversion rates and thus be game-changing with regard to industrial steroid production efficiency.

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.

Hydrogen is an important building block in the chemical industry, but over the last decades, several attempts were also made to develop hydrogen as an energy carrier, for example, for fuel cell technology (electricity, mobility, heating). While this had limited success in the past, there is a renewed push for a systematic change to hydrogen as a major energy carrier. This shift is mainly driven by the widely accepted understanding that the observed harsh effects of climate change must be to a great deal connected to the burning of fossil energy carriers. We introduce a basic concept for producing hydrogen from water using natural photosynthesis applying the toolbox of white biotechnology. We term this "White hydrogen." Water is split to electrons, protons and oxygen by photosystem 2 and hydrogen is subsequently formed. There are basically two approaches to achieve hydrogen formation, both using microbial whole cell biocatalysts. On the one hand, in biophotolysis, electrons liberated in the water oxidizing reaction are delivered via the photosystem(s) to a hydrogenase without a detour through central metabolism, but directly via a redox cofactor like NAD(P)H or ferredoxin. Oxygen is formed in the same cell as a by-product of the water splitting reaction and, as it is the case for hydrogen itself, must be separated. On the other hand, in biophotovoltaics, the photosynthetic electron transport chain is connected to a solid state electrode (anode), driving the reduction of protons to hydrogen on the cathode of a microbial electrolysis cell. Thereby, the formation of oxygen and hydrogen occurs in different reaction chambers, facilitating product recovery. While we expect the future energy mix to comprise bulk hydrogen produced in centralized facilities, for example, via electrolysis driven by electricity derived from photovoltaics or wind power plants, we believe that low-cost and less-resource-intensive solutions will make an important contribution in decentralized, autonomous facilities and applications. This could be smallscale production units for white hydrogen up to a few hundred kg per year that might be directly connected with hydrogen use after short-term storage circumventing complex logistics for large-scale transport and storage. For this purpose, continuous reaction formats are required, for example, the use of phototrophic biofilms as biocatalysts for the production of white hydrogen at biomass concentrations and light supply tuned for optimal hydrogen production efficiency. This chapter presents and discusses the frame, status, potential and challenges of these two approaches and proposes a concept for the integrated development of the molecular machinery, the biocatalysts and suitable reaction and process formats.

6-hydroxyhexanoic acid (6HA) represents a polymer building block for the biodegradable polymer polycaprolactone. Alternatively to energy- and emission-intensive multistep chemical synthesis, it can be synthesized directly from cyclohexane in one step by recombinant Pseudomonas taiwanensis harboring a 4-step enzymatic cascade without the accumulation of any intermediate. In the present work, we performed a physiological characterization of this strain in different growth media and evaluated the resulting whole-cell activities. RB and M9* media led to reduced gluconate accumulation from glucose compared to M9 medium and allowed specific activities up to 37.5 ± 0.4 U g CDW −1 for 6HA synthesis. However, 50% of the specific activity was lost within 1 h in metabolically active resting cells, specifying growing cells, or induced resting cells as favored options for long-term biotransformation. Furthermore, the whole-cell biocatalyst was evaluated in a stirred-tank bioreactor setup with a continuous cyclohexane supply via the gas phase. At cyclohexane feed rates of 0.276 and 1.626 mmol min −1 L −1 , whole-cell biotransformation occurred at first-order and zero-order rates, respectively. A final 6HA concentration of 25 mM (3.3 g L −1 ) and a specific product yield of 0.4 g g CDW −1 were achieved with the higher feed rate. Product inhibition and substrate toxification were identified as critical factors limiting biocatalytic performance. Future research efforts on these factors and the precise adjustment of the cyclohexane feed combined with an in situ product removal strategy are discussed as promising strategies to enhance biocatalyst durability and product titer and thus to enable the development of a sustainable multistep whole-cell process.