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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.

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.

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.

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.

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.

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 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 gCDW-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. Further, 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 0.221 and 1.084 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 gCDW-1 were achieved with the higher feed rate. Substrate limitation, product inhibition, and toxification by cyclohexane were identified as critical factors limiting biocatalytic performance. Future research efforts on these factors and 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.

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. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO‐biocatalyst formats, i.e., 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 to 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, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an 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.

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, aut

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.

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