Nils Averesch

All Publications

• Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities. ACS synthetic biology Rothschild, L. J., Averesch, N. J., Strychalski, E. A., Moser, F., Glass, J. I., Cruz Perez, R., Yekinni, I. O., Rothschild-Mancinelli, B., Roberts Kingman, G. A., Wu, F., Waeterschoot, J., Ioannou, I. A., Jewett, M. C., Liu, A. P., Noireaux, V., Sorenson, C., Adamala, K. P. 2024

  Abstract

  The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

  View details for DOI 10.1021/acssynbio.3c00724

  View details for PubMedID 38530077

• Space bioprocess engineering as a potential catalyst for sustainability NATURE SUSTAINABILITY Vengerova, G., Lipsky, I., Hutchinson, G. A., Averesch, N. H., Berliner, A. J. 2024; 7 (3): 238-246

  View details for DOI 10.1038/s41893-024-01305-x

  View details for Web of Science ID 001189384800013

• Microbial biomanufacturing for space-exploration-what to take and when to make. Nature communications Averesch, N. J., Berliner, A. J., Nangle, S. N., Zezulka, S., Vengerova, G. L., Ho, D., Casale, C. A., Lehner, B. A., Snyder, J. E., Clark, K. B., Dartnell, L. R., Criddle, C. S., Arkin, A. P. 2023; 14 (1): 2311

  Abstract

  As renewed interest in human space-exploration intensifies, a coherent and modernized strategy for mission design and planning has become increasingly crucial. Biotechnology has emerged as a promising approach to increase resilience, flexibility, and efficiency of missions, by virtue of its ability to effectively utilize in situ resources and reclaim resources from waste streams. Here we outline four primary mission-classes on Moon and Mars that drive a staged and accretive biomanufacturing strategy. Each class requires a unique approach to integrate biomanufacturing into the existing mission-architecture and so faces unique challenges in technology development. These challenges stem directly from the resources available in a given mission-class-the degree to which feedstocks are derived from cargo and in situ resources-and the degree to which loop-closure is necessary. As mission duration and distance from Earth increase, the benefits of specialized, sustainable biomanufacturing processes also increase. Consequentially, we define specific design-scenarios and quantify the usefulness of in-space biomanufacturing, to guide techno-economics of space-missions. Especially materials emerged as a potentially pivotal target for biomanufacturing with large impact on up-mass cost. Subsequently, we outline the processes needed for development, testing, and deployment of requisite technologies. As space-related technology development often does, these advancements are likely to have profound implications for the creation of a resilient circular bioeconomy on Earth.

  View details for DOI 10.1038/s41467-023-37910-1

  View details for PubMedID 37085475

  View details for PubMedCentralID PMC10121718

• Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms. Nature communications Santomartino, R., Averesch, N. J., Bhuiyan, M., Cockell, C. S., Colangelo, J., Gumulya, Y., Lehner, B., Lopez-Ayala, I., McMahon, S., Mohanty, A., Santa Maria, S. R., Urbaniak, C., Volger, R., Yang, J., Zea, L. 2023; 14 (1): 1391

  Abstract

  Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

  View details for DOI 10.1038/s41467-023-37070-2

  View details for PubMedID 36944638

• Cultivation of the Dematiaceous Fungus Cladosporium sphaerospermum Aboard the International Space Station and Effects of Ionizing Radiation. Frontiers in microbiology Averesch, N. J., Shunk, G. K., Kern, C. 2022; 13: 877625

  Abstract

  In Space, cosmic radiation is a strong, ubiquitous form of energy with constant flux, and the ability to harness it could greatly enhance the energy-autonomy of expeditions across the solar system. At the same time, radiation is the greatest permanent health risk for humans venturing into deep space. To protect astronauts beyond Earth's magnetosphere, advanced shielding against ionizing as well as non-ionizing radiation is highly sought after. In search of innovative solutions to these challenges, biotechnology appeals with suitability for in situ resource utilization (ISRU), self-regeneration, and adaptability. Where other organisms fail, certain microscopic fungi thrive in high-radiation environments on Earth, showing high radioresistance. The adaptation of some of these molds to areas, such as the Chernobyl Exclusion Zone has coined the terms positive "radiotropism" and "radiotrophy", reflecting the affinity to and stimulation by radiation, and sometimes even enhanced growth under ionizing conditions. These abilities may be mediated by the pigment melanin, many forms of which also have radioprotective properties. The expectation is that these capabilities are extendable to radiation in space. To study its growth in space, an experiment cultivating Cladosporium sphaerospermum Penzig ATCC 11289 aboard the International Space Station (ISS) was conducted while monitoring radiation beneath the formed biomass in comparison to a no-growth negative control. A qualitative growth advantage in space was observable. Quantitatively, a 1.21 ± 0.37-times higher growth rate than in the ground control was determined, which might indicate a radioadaptive response to space radiation. In addition, a reduction in radiation compared to the negative control was discernable, which is potentially attributable to the fungal biomass.

  View details for DOI 10.3389/fmicb.2022.877625

  View details for PubMedID 35865919

• Space bioprocess engineering on the horizon Communications Engineering Berliner, A. J., Lipsky, I., Ho, D., Hilzinger, J. M., Vengerova, G., Makrygiorgos, G., McNulty, M. J., Yates, K., Averesch, N. J., Cockell, C. S., Wallentine, T., Seefeldt, L. C., Criddle, C. S., Nandi, S., McDonald, K. A., Menezes, A. A., Mesbah, A., Arkin , A. P. 2022; 1

  View details for DOI 10.1038/s44172-022-00012-9

• Towards a Biomanufactory on Mars FRONTIERS IN ASTRONOMY AND SPACE SCIENCES Berliner, A. J., Hilzinger, J. M., Abel, A. J., McNulty, M. J., Makrygiorgos, G., Averesch, N. H., Sen Gupta, S., Benvenuti, A., Caddell, D. F., Cestellos-Blanco, S., Doloman, A., Friedline, S., Ho, D., Gu, W., Hill, A., Kusuma, P., Lipsky, I., Mirkovic, M., Meraz, J., Pane, V., Sander, K. B., Shi, F., Skerker, J. M., Styer, A., Valgardson, K., Wetmore, K., Woo, S., Xiong, Y., Yates, K., Zhang, C., Zhen, S., Bugbee, B., Clark, D. S., Coleman-Derr, D., Mesbah, A., Nandi, S., Waymouth, R. M., Yang, P., Criddle, C. S., McDonald, K. A., Seefeldt, L. C., Menezes, A. A., Arkin, A. P. 2021; 8

  View details for DOI 10.3389/fspas.2021.711550

  View details for Web of Science ID 000680691100001

• Choice of Microbial System for In-Situ Resource Utilization on Mars FRONTIERS IN ASTRONOMY AND SPACE SCIENCES Averesch, N. 2021; 8

  View details for DOI 10.3389/fspas.2021.700370

  View details for Web of Science ID 000673223100001

• Anodic electro-fermentation: Empowering anaerobic production processes via anodic respiration. Biotechnology advances Vassilev, I., Averesch, N. J., Ledezma, P., Kokko, M. 2021: 107728

  Abstract

  In nature as well as in industrial microbiology, all microorganisms need to achieve redox balance. Their redox state and energy conservation highly depend on the availability of a terminal electron acceptor, for example oxygen in aerobic production processes. Under anaerobic conditions in the absence of an electron acceptor, redox balance is achieved via the production of reduced carbon-compounds (fermentation). An alternative strategy to artificially stabilize microbial redox and energy state is the use of anodic electro-fermentation (AEF). This emerging biotechnology empowers respiration under anaerobic conditions using the anode of a bioelectrochemical system as an undepletable terminal electron acceptor. Electrochemical control of redox metabolism and energy conservation via AEF can steer the carbon metabolism towards a product of interest and avoid the need for continuous and cost-inefficient supply of oxygen as well as the production of mixed reduced by-products, as is the case in aerobic production and fermentation processes. The great challenge for AEF is to establish efficient extracellular electron transfer (EET) from the microbe to the anode and link it to central carbon metabolism to enhance the synthesis of a target product. This article reviews the advantages and challenges of AEF, EET mechanisms, microbial energy gain, and discusses the rational choice of substrate-product couple as well as the choice of microbial catalyst. Besides, it discusses the potential of the industrial model-organism Bacillus subtilis as a promising candidate for AEF, which has not been yet considered for such an application. This prospective review contributes to a better understanding of how industrial microbiology can benefit from AEF and analyses key-factors required to successfully implement AEF processes. Overall, this work aims to advance the young research field especially by critically revisiting the fundamental aspects of AEF.

  View details for DOI 10.1016/j.biotechadv.2021.107728

  View details for PubMedID 33705913

• Editorial: Biotechnological Production and Conversion of Aromatic Compounds and Natural Products. Frontiers in bioengineering and biotechnology Averesch, N. J., Kayser, O. n. 2020; 8: 646

  View details for DOI 10.3389/fbioe.2020.00646

  View details for PubMedID 32637405

  View details for PubMedCentralID PMC7318798

• Metabolic engineering of Bacillus subtilis for production of para-aminobenzoic acid - unexpected importance of carbon source is an advantage for space application MICROBIAL BIOTECHNOLOGY Averesch, N. H., Rothschild, L. J. 2019; 12 (4): 703–14

  Abstract

  High-strength polymers, such as aramid fibres, are important materials in space technology. To obtain these materials in remote locations, such as Mars, biological production is of interest. The aromatic polymer precursor para-aminobenzoic acid (pABA) can be derived from the shikimate pathway through metabolic engineering of Bacillus subtilis, an organism suited for space synthetic biology. Our engineering strategy included repair of the defective indole-3-glycerol phosphate synthase (trpC), knockout of one chorismate mutase isozyme (aroH) and overexpression of the aminodeoxychorismate synthase (pabAB) and aminodeoxychorismate lyase (pabC) from the bacteria Corynebacterium callunae and Xenorhabdus bovienii respectively. Further, a fusion-protein enzyme (pabABC) was created for channelling of the carbon flux. Using adaptive evolution, mutants of the production strain, able to metabolize xylose, were created, to explore and compare pABA production capacity from different carbon sources. Rather than the efficiency of the substrate or performance of the biochemical pathway, the product toxicity, which was strongly dependent on the pH, appeared to be the overall limiting factor. The highest titre achieved in shake flasks was 3.22 g l-1 with a carbon yield of 12.4% [C-mol/C-mol] from an amino sugar. This promises suitability of the system for in situ resource utilization (ISRU) in space biotechnology, where feedstocks that can be derived from cyanobacterial cell lysate play a role.

  View details for DOI 10.1111/1751-7915.13403

  View details for Web of Science ID 000473648300011

  View details for PubMedID 30980511

  View details for PubMedCentralID PMC6559200

• Metabolic Network Analysis of Microbial Methane Utilization for Biomass Formation and Upgrading to Bio-Fuels FRONTIERS IN ENERGY RESEARCH Averesch, N. H., Kracke, F. 2018; 6

  View details for DOI 10.3389/fenrg.2018.00106

  View details for Web of Science ID 000447328000001

• Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY Averesch, N. H., Kroemer, J. O. 2018; 6: 32

  Abstract

  The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

  View details for DOI 10.3389/fbioe.2018.00032

  View details for Web of Science ID 000440256500001

  View details for PubMedID 29632862

  View details for PubMedCentralID PMC5879953

• Toward Synthetic Biology Strategies for Adipic Acid Production: An in Silico Tool for Combined Thermodynamics and Stoichiometric Analysis of Metabolic Networks ACS SYNTHETIC BIOLOGY Averesch, N. H., Martinez, V. S., Nielsen, L. K., Kromer, J. O. 2018; 7 (2): 490-509

  Abstract

  Adipic acid, a nylon-6,6 precursor, has recently gained popularity in synthetic biology. Here, 16 different production routes to adipic acid were evaluated using a novel tool for network-embedded thermodynamic analysis of elementary flux modes. The tool distinguishes between thermodynamically feasible and infeasible modes under determined metabolite concentrations, allowing the thermodynamic feasibility of theoretical yields to be assessed. Further, patterns that always caused infeasible flux distributions were identified, which will aid the development of tailored strain design. A review of cellular efflux mechanisms revealed that significant accumulation of extracellular product is only possible if coupled with ATP hydrolysis. A stoichiometric analysis demonstrated that the maximum theoretical product carbon yield heavily depends on the metabolic route, ranging from 32 to 99% on glucose and/or palmitate in Escherichia coli and Saccharomyces cerevisiae metabolic models. Equally important, metabolite concentrations appeared to be thermodynamically restricted in several pathways. Consequently, the number of thermodynamically feasible flux distributions was reduced, in some cases even rendering whole pathways infeasible, highlighting the importance of pathway choice. Only routes based on the shikimate pathway were thermodynamically favorable over a large concentration and pH range. The low pH capability of S. cerevisiae shifted the thermodynamic equilibrium of some pathways toward product formation. One identified infeasible-pattern revealed that the reversibility of the mitochondrial malate dehydrogenase contradicted the current state of knowledge, which imposes a major restriction on the metabolism of S. cerevisiae. Finally, the evaluation of industrially relevant constraints revealed that two shikimate pathway-based routes in E. coli were the most robust.

  View details for DOI 10.1021/acssynbio.7b00304

  View details for Web of Science ID 000426012600021

  View details for PubMedID 29237121

• Enhanced production of para-hydroxybenzoic acid by genetically engineered Saccharomyces cerevisiae BIOPROCESS AND BIOSYSTEMS ENGINEERING Averesch, N. H., Prima, A., Kroemer, J. O. 2017; 40 (8): 1283-1289

  Abstract

  Saccharomyces cerevisiae is a popular organism for metabolic engineering; however, studies aiming at over-production of bio-replacement precursors for the chemical industry often fail to overcome proof-of-concept stage. When intending to show real industrial attractiveness, the challenge is twofold: formation of the target compound must be increased, while minimizing the formation of side and by-products to maximize titer, rate and yield. To tackle these, the metabolism of the organism, as well as the parameters of the process, need to be optimized. Addressing both we show that S. cerevisiae is well-suited for over-production of aromatic compounds, which are valuable in chemical industry and are particularly useful in space technology. Specifically, a strain engineered to accumulate chorismate was optimized for formation of para-hydroxybenzoic acid. Then a fed-batch bioreactor process was developed, which delivered a final titer of 2.9 g/L, a maximum rate of 18.625 mgpHBA/(gCDW × h) and carbon-yields of up to 3.1 mgpHBA/gglucose.

  View details for DOI 10.1007/s00449-017-1785-z

  View details for Web of Science ID 000405490000013

  View details for PubMedID 28528488

• Quantitative analysis of aromatics for synthetic biology using liquid chromatography Biotechnology Journal Lai, B., Plan, M. R., Averesch, N. J., Yu, S., Kracke, F., Lekieffre, N., Bydder, S., Hodson, M. P., Winter, G., Krömer, J. O. 2017; 12 (1)

  Abstract

  The replacement of petrochemical aromatics with bio-based molecules is a key area of current biotechnology research. To date, a small number of aromatics have been produced by recombinant bacteria in laboratory scale while industrial production still requires further strain development. While each study includes some distinct analytical methodology to quantify certain aromatics, a method that can reliably quantify a great number of aromatic products and relevant pathway intermediates is needed to accelerate strain development. In this study, we developed a robust reverse phase high performance liquid chromatography method to quantify a wide range of aromatic metabolites present in host microorganisms using the shikimate pathway, which is the major metabolic pathway for biosynthesis of aromatics. Twenty-three metabolites can be quantified precisely with the optimized method using standard HPLC equipment and UV detection, with the mobile phase used for chromatography also compatible with mass spectrometry (MS). The limit of quantification/detection is as low as 10-10 to 10-13 mol, respectively, which makes this method feasible for quantification of intracellular metabolites. This method covers most metabolic routes for aromatics biosynthesis, it is inexpensive, robust, simple, precise and sensitive, and has been demonstrated on cell extracts from S. cerevisiae genetically engineered to overproduce aromatics.

  View details for DOI 10.1002/biot.201600269

• Discrimination of wild types and hybrids of Duboisia myoporoides and Duboisia leichhardtii at different growth stages using H-1 NMR-based metabolite profiling and tropane alkaloids-targeted HPLC-MS analysis PHYTOCHEMISTRY Ullrich, S., Averesch, N. H., Castellanos, L., Choi, Y., Rothauer, A., Kayser, O. 2016; 131: 44-56

  Abstract

  Duboisia species, which belong to the family of Solanaceae, are commercially cultivated in large scale, as they are main source of the pharmaceutically-used active compound scopolamine. In this study, 1H NMR-based metabolite profiling linking primary with secondary metabolism and additional quantification via HPCL-MS with special focus on the tropane alkaloids were applied to compare leaf and root extracts of three wild types and two hybrids of Duboisia myoporoides and D. leichhardtii at different developmental stages grown under controlled conditions in climate chambers and under agricultural field plantation. Based on the leaf extracts, a clear distinction between the Duboisia hybrids and the wild types Duboisia myoporoides and D. leichhardtii using principal component analysis of 1H NMR data was observed. The average content in scopolamine in the hybrids of Duboisia cultivated in climate chambers increased significantly from month 3-6 after potting of the rooted cuttings, however not so for the examined wild types. The Duboisia hybrids grown in climate chambers showed higher growth and contained more sugars and amino acids than Duboisia hybrids grown in the field, which in contrast showed an enhanced flux towards tropane alkaloids as well as flavonoids. For a more detailed analysis of tropane alkaloids, an appropriate HPLC-MS method was developed and validated. The measurements revealed large differences in the alkaloid pattern within the different genotypes under investigation, especially regarding the last enzymatic step, the conversion from hyoscamine to scopolamine by the hyoscyamine 6β-hydroxylase. Scopolamine was found in highest concentrations in Duboisia hybrids (20.04 ± 4.05 and 17.82 ± 3.52 mg/g dry wt) followed by Duboisia myoporoides (12.71 ± 2.55 mg/g dry wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38 ± 0.59 and 5.09 ± 1.24 mg/g dry wt) with hyoscyamine being the predominant alkaloid.

  View details for DOI 10.1016/j.phytochem.2016.08.008

  View details for Web of Science ID 000385692000005

  View details for PubMedID 27567452

• Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae MICROBIAL CELL FACTORIES Averesch, N. H., Winter, G., Kromer, J. O. 2016; 15: 89

  Abstract

  Biological production of the aromatic compound para-aminobenzoic acid (pABA) is of great interest to the chemical industry. Besides its application in pharmacy and as crosslinking agent for resins and dyes pABA is a potential precursor for the high-volume aromatic feedstocks terephthalic acid and para-phenylenediamine. The yeast Saccharomyces cerevisiae synthesises pABA in the shikimate pathway: Outgoing from the central shikimate pathway intermediate chorismate, pABA is formed in two enzyme-catalysed steps, encoded by the genes ABZ1 and ABZ2. In this study S. cerevisiae metabolism was genetically engineered for the overproduction of pABA. Using in silico metabolic modelling an observed impact of carbon-source on product yield was investigated and exploited to optimize production.A strain that incorporated the feedback resistant ARO4 (K229L) and deletions in the ARO7 and TRP3 genes, in order to channel flux to chorismate, was used to screen different ABZ1 and ABZ2 genes for pABA production. In glucose based shake-flaks fermentations the highest titer (600 µM) was reached when over-expressing the ABZ1 and ABZ2 genes from the wine yeast strains AWRI1631 and QA23, respectively. In silico metabolic modelling indicated a metabolic advantage for pABA production on glycerol and combined glycerol-ethanol carbon-sources. This was confirmed experimentally, the empirical ideal glycerol to ethanol uptake ratios of 1:2-2:1 correlated with the model. A (13)C tracer experiment determined that up to 32% of the produced pABA originated from glycerol. Finally, in fed-batch bioreactor experiments pABA titers of 1.57 mM (215 mg/L) and carbon yields of 2.64% could be achieved.In this study a combination of genetic engineering and in silico modelling has proven to be a complete and advantageous approach to increase pABA production. Especially the enzymes that catalyse the last two steps towards product formation appeared to be crucial to direct flux to pABA. A stoichiometric model for carbon-utilization proved useful to design carbon-source composition, leading to increased pABA production. The reported pABA concentrations and yields are, to date, the highest in S. cerevisiae and the second highest in a microbial production system, underlining the great potential of yeast as a cell factory for renewable aromatic feedstocks.

  View details for DOI 10.1186/s12934-016-0485-8

  View details for Web of Science ID 000377167300001

  View details for PubMedID 27230236

  View details for PubMedCentralID PMC4882779

• Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae METABOLIC ENGINEERING Williams, T. C., Averesch, N. H., Winter, G., Plan, M. R., Vickers, C. E., Nielsen, L. K., Kroemer, J. O. 2015; 29: 124-134

  Abstract

  Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

  View details for DOI 10.1016/j.ymben.2015.03.008

  View details for Web of Science ID 000354123900013

  View details for PubMedID 25792511

• Tailoring strain construction strategies for muconic acid production inS. cerevisiaeandE. coli. Metabolic engineering communications Averesch, N. J., Kromer, J. O. 2014; 1: 19-28

  Abstract

  There is currently a strong interest to derive the biological precursor cis,cis-muconic acid from shikimate pathway-branches to develop a biological replacement for adipic acid. Pioneered by the Frost laboratory this concept has regained interest: Recent approaches (Boles, Alper, Yan) however suffer from low product titres. Here an in silico comparison of all strain construction strategies was conducted to highlight stoichiometric optimizations. Using elementary mode analysis new knock-out strategies were determined in Saccharomyces cerevisiae and Escherichia coli. The strain construction strategies are unique to each pathway-branch and organism, allowing significantly different maximum and minimum yields. The maximum theoretical product carbon yields on glucose ranged from 86% (dehydroshikimate-branch) to 69% (anthranilate-branch). In most cases a coupling of product formation to growth was possible. Especially in S. cerevisiae chorismate-routes a minimum yield constraint of 46.9% could be reached. The knock-out targets are non-obvious, and not-transferable, highlighting the importance of tailored strain construction strategies.

  View details for DOI 10.1016/j.meteno.2014.09.001

  View details for PubMedID 34150501

• In vivo instability of chorismate causes substrate loss during fermentative production of aromatics YEAST Winter, G., Averesch, N. H., Nunez-Bernal, D., Kroemer, J. O. 2014; 31 (9): 333-341

  Abstract

  Metabolic engineering of microbial strains to produce aromatic compounds deriving from the shikimate pathway is of great interest to the chemical industry as a more sustainable alternative for feedstock production. Chorismate is a significant intermediate in the shikimate pathway. In this study, the formation of phenylalanine and phenylpyruvate as by-products in strains engineered downstream of the chorismate node for increased aromatic production was explored in yeast fermentations. Tracer experiments showed that these compounds are synthesized de novo during fermentation, under conditions in which their synthesis was genetically blocked. Chorismate stability evaluation, as well as deletion mutation analysis throughout the phenylalanine biosynthesis pathway, suggested that this synthesis was a result of intracellular, non-enzymatic rearrangement of chorismate to phenylpyruvate via prephenate, which was followed by enzymatic transamination of phenylpyruvate to form phenylalanine. These results not only aid in the development of strain-engineering strategies to avoid the accumulation of by-products during fermentations aimed at increased aromatics production, but also deepen our understanding of yeast metabolism.

  View details for DOI 10.1002/yea.3025

  View details for Web of Science ID 000342918800002

  View details for PubMedID 24981409

• Assessing Heterologous Expression of Hyoscyamine 6 beta-Hydroxylase - a Feasibility Study Averesch, N. H., Kayser, O., Duc, N. M., Elfahmi, Wirasutisna, K. R., Sukrasno, Suganda, A. G., Abe, Petersen, M., Insanu, Hartati, R., Pramastya, H., Julianti, E., Fisheri, N. ELSEVIER SCIENCE BV. 2014: 69-78

  View details for DOI 10.1016/j.proche.2014.12.008

  View details for Web of Science ID 000353466400007

• Production of aromatics in Saccharomyces cerevisiae-A feasibility study JOURNAL OF BIOTECHNOLOGY Kroemer, J. O., Nunez-Bernal, D., Averesch, N. H., Hampe, J., Varela, J., Varela, C. 2013; 163 (2): 184-193

  Abstract

  Aromatics are amongst the most important bulk feedstocks for the chemical industry, however, no viable bioprocess exists today and production is still dependent on petro-chemistry. In this article the production of aromatic precursors such as p-hydroxybenzoic acid (PHBA) and p-amino benzoic acid (PABA) in Saccharomyces cerevisiae was evaluated using metabolic network analysis. Theoretical mass yields for PHBA and for PABA obtained by metabolic network analysis were 0.58 and 0.53 g g(glucose)⁻¹, respectively. A major setback for microbial production of aromatics is the high toxicity of the products. Therefore, PHBA and PABA toxicity was evaluated in S. cerevisiae. Minimal inhibitory concentrations of 38.3 g L⁻¹ for PHBA and 0.62 g L⁻¹ for PABA were observed. However, PABA toxicity could be alleviated in adaptation experiments. Finally, metabolic engineering was used to create proof of principle first generation strains of S. cerevisiae. Overall accumulation of 650 μM PHBA and 250 μM PABA could be achieved.

  View details for DOI 10.1016/j.jbiotec.2012.04.014

  View details for Web of Science ID 000313738700012

  View details for PubMedID 22579724

• Organosoluble enzyme conjugates with poly(2-oxazoline)s via pyromellitic acid dianhydride JOURNAL OF BIOTECHNOLOGY Konieczny, S., Fik, C. P., Averesch, N. H., Tiller, J. C. 2012; 159 (3): 195-203

  Abstract

  The use of enzymes in organic solvents offers a great opportunity for the synthesis of complex organic compounds and is therefore in focus of current research. In this work we describe the synthesis of poly(2-methyl-1,3-oxazoline) (PMOx) and poly(2-ethyl-1,3-oxazoline) (PEtOx) enzyme conjugates with hen-egg white lysozyme, RNase A and α-chymotrypsin using a new coupling technique. The POXylation was carried out reacting pyromellitic acid dianhydride subsequently with ethylenediamine terminated POx and then with the NH₂-groups of the respective enzymes. Upon conjugation with the polymers, RNase A and lysozyme became fully soluble in DMF (1.4 mg/ml). These are the first examples of fully POXylated proteins, which become organosoluble. The synthesized enzyme conjugates were characterized by SDS-PAGE, isoelectric focusing, dynamic light scattering and size exclusion chromatography, which all indicated the full POXylation of the enzymes. The modified enzymes even partly retained their activity in water. With α-chymotrypsin as example we could demonstrate that the molecular weight of the attached polymer significantly influences the activity.

  View details for DOI 10.1016/j.jbiotec.2012.01.016

  View details for Web of Science ID 000303935000010

  View details for PubMedID 22306109