Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE).

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.

Automated in vivo enzyme engineering accelerates biocatalyst optimization.

Achieving cost-competitive bio-based processes requires development of stable and selective biocatalysts. Their realization through in vitro enzyme characterization and engineering is mostly low throughput and labor-intensive. Therefore, strategies for increasing throughput while diminishing manual labor are gaining momentum, such as in vivo screening and evolution campaigns. Computational tools like machine learning further support enzyme engineering efforts by widening the explorable design space. Here, we propose an integrated solution to enzyme engineering challenges whereby ML-guided, automated workflows (including library generation, implementation of hypermutation systems, adapted laboratory evolution, and in vivo growth-coupled selection) could be realized to accelerate pipelines towards superior biocatalysts.

Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

The demand for highly robust and metabolically versatile microbes is of utmost importance for replacing fossil-based processes with biotechnological ones. Such an example is the implementation of Paenibacillus polymyxa DSM 365 as a novel platform organism for the production of value-added products such as 2,3-butanediol or exopolysaccharides. For this, a complete genome sequence is the first requirement towards further developing this host towards a microbial chassis. A genome sequencing project has just been reported for P. polymyxa DSM 365 showing a size of 5,788,318 bp with a total of 47 contigs. Herein, we report the first complete genome sequence of P. polymyxa DSM 365, which consists of 5,889,536 bp with 45 RNAs, 106 tRNAs, 5,370 coding sequences and an average GC content of 45.6%, resulting in a closed genome of P. polymyxa 365. The additional nucleotide data revealed a novel NRPS synthetase that may contribute to the production of tridecaptin. Building on these findings, we initiated the top-down construction of a chassis variant of P. polymyxa. In the first stage, single knock-out mutants of non-essential genomic regions were created and evaluated for their biological fitness. As a result, two out of 18 variants showed impaired growth. The remaining deletion mutants were combined in two genome-reduced P. polymyxa variants which either lack the production of endogenous biosynthetic gene clusters (GR1) or non-essential genomic regions including the insertion sequence ISPap1 (GR2), with a decrease of the native genome of 3.0% and 0.6%, respectively. Both variants, GR1 and GR2, showed identical growth characteristics to the wild-type. Endpoint titers of 2,3-butanediol and EPS production were also unaffected, validating these genome-reduced strains as suitable for further genetic engineering.

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h-1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Robotic workflows for automated long-term adaptive laboratory evolution: improving ethanol utilization by Corynebacterium glutamicum.

BACKGROUND: Adaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation. RESULTS: Here, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW-1 h-1 and a growth rate of 0.15 ± 0.01 h-1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements. CONCLUSIONS: The established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

BACKGROUND: Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. RESULTS: In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. CONCLUSIONS: This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

Achieving net zero CO2 emission in the biobased production of reduced platform chemicals using defined co-feeding of methanol.

Next-generation bioprocesses of a future bio-based economy will rely on a flexible mix of readily available feedstocks. Renewable energy can be used to generate sustainable CO2-derived substrates. Metabolic engineering already enables the functional implementation of different pathways for the assimilation of C1 substrates in various microorganisms. In addition to feedstocks, the benchmark for all future bioprocesses will be sustainability, including the avoidance of CO2 emissions. Here we review recent advances in the utilization of C1-compounds from different perspectives, considering both strain and bioprocess engineering technologies. In particular, we evaluate methanol as a co-feed for enabling the CO2 emission-free production of acetyl-CoA-derived compounds. The possible metabolic strategies are analyzed using stoichiometric modeling combined with thermodynamic analysis and prospects for industrial-scale implementation are discussed.

Introducing molasses as an alternative feedstock into itaconate production using Ustilago sp.

In this work, we established an efficient process for the production of itaconate from the regionally sourced industrial side-stream molasses using Ustilago cynodontis and Ustilago maydis. While being relatively cheap and more environmentally friendly than refined sugars, there are some major challenges to overcome when working with molasses. Some of those challenges are a high nitrogen load, unknown impurities in the feedstock, and high amounts of ill-favoured carbon sources, such as sucrose or lactate. We could show that the activity of the sucrose-hydrolysing enzyme invertase plays a crucial role in the efficiency of the process and that the fructose utilisation differs between the two strains used in this work. Thus, with a higher invertase activity, the ability to convert fructose into the desired product itaconate, and an overall higher tolerance towards undesired substances in molasses, U. maydis is better equipped for the process on the alternative feedstock molasses than U. cynodontis. The established process with U. maydis reached competitive yields of up to 0.38 g g-1 and a titre of more than 37 g L-1. This shows that an efficient and cost-effective itaconate production process is generally feasible using U. maydis, which has the potential to greatly increase the sustainability of industrial itaconate production.

Holistic Approach to Process Design and Scale-Up for Itaconic Acid Production from Crude Substrates.

Bio-based bulk chemicals such as carboxylic acids continue to struggle to compete with their fossil counterparts on an economic basis. One possibility to improve the economic feasibility is the use of crude substrates in biorefineries. However, impurities in these substrates pose challenges in fermentation and purification, requiring interdisciplinary research. This work demonstrates a holistic approach to biorefinery process development, using itaconic acid production on thick juice based on sugar beets with Ustilago sp. as an example. A conceptual process design with data from artificially prepared solutions and literature data from fermentation on glucose guides the simultaneous development of the upstream and downstream processes up to a 100 L scale. Techno-economic analysis reveals substrate consumption as the main constituent of production costs and therefore, the product yield is the driver of process economics. Aligning pH-adjusting agents in the fermentation and the downstream process is a central lever for product recovery. Experiments show that fermentation can be transferred from glucose to thick juice by changing the feeding profile. In downstream processing, an additional decolorization step is necessary to remove impurities accompanying the crude substrate. Moreover, we observe an increased use of pH-adjusting agents compared to process simulations.

Discovery of novel amino acid production traits by evolution of synthetic co-cultures.

BACKGROUND: Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities. RESULTS: Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain. CONCLUSION: Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS , is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS . In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial kS estimation, sl-MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl-MSCC lays the foundation for strain-specific kS estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.

Characterization and engineering of branched short-chain dicarboxylate metabolism in Pseudomonas reveals resistance to fungal 2-hydroxyparaconate.

In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications.

Communities of Niche-optimized Strains (CoNoS) - Design and creation of stable, genome-reduced co-cultures.

Current bioprocesses for production of value-added compounds are mainly based on pure cultures that are composed of rationally engineered strains of model organisms with versatile metabolic capacities. However, in the comparably well-defined environment of a bioreactor, metabolic flexibility provided by various highly abundant biosynthetic enzymes is much less required and results in suboptimal use of carbon and energy sources for compound production. In nature, non-model organisms have frequently evolved in communities where genome-reduced, auxotrophic strains cross-feed each other, suggesting that there must be a significant advantage compared to growth without cooperation. To prove this, we started to create and study synthetic communities of niche-optimized strains (CoNoS) that consists of two strains of the same species Corynebacterium glutamicum that are mutually dependent on one amino acid. We used both the wild-type and the genome-reduced C1* chassis for introducing selected amino acid auxotrophies, each based on complete deletion of all required biosynthetic genes. The best candidate strains were used to establish several stably growing CoNoS that were further characterized and optimized by metabolic modelling, microfluidic experiments and rational metabolic engineering to improve amino acid production and exchange. Finally, the engineered CoNoS consisting of an l-leucine and l-arginine auxotroph showed a specific growth rate equivalent to 83% of the wild type in monoculture, making it the fastest co-culture of two auxotrophic C. glutamicum strains to date. Overall, our results are a first promising step towards establishing improved biobased production of value-added compounds using the CoNoS approach.

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments.

BACKGROUND: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. RESULTS: We present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. CONCLUSIONS: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

bletl - A Python package for integrating BioLector microcultivation devices in the Design-Build-Test-Learn cycle.

Microbioreactor (MBR) devices have emerged as powerful cultivation tools for tasks of microbial phenotyping and bioprocess characterization and provide a wealth of online process data in a highly parallelized manner. Such datasets are difficult to interpret in short time by manual workflows. In this study, we present the Python package bletl and show how it enables robust data analyses and the application of machine learning techniques without tedious data parsing and preprocessing. bletl reads raw result files from BioLector I, II and Pro devices to make all the contained information available to Python-based data analysis workflows. Together with standard tooling from the Python scientific computing ecosystem, interactive visualizations and spline-based derivative calculations can be performed. Additionally, we present a new method for unbiased quantification of time-variable specific growth rate µ ⃗ t based on unsupervised switchpoint detection with Student-t distributed random walks. With an adequate calibration model, this method enables practitioners to quantify time-variable growth rate with Bayesian uncertainty quantification and automatically detect switch-points that indicate relevant metabolic changes. Finally, we show how time series feature extraction enables the application of machine learning methods to MBR data, resulting in unsupervised phenotype characterization. As an example, Neighbor Embedding (t-SNE) is performed to visualize datasets comprising a variety of growth/DO/pH phenotypes.

Biosensor-based growth-coupling and spatial separation as an evolution strategy to improve small molecule production of Corynebacterium glutamicum.

Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of 'cheater' strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.