More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as medical component. So far, only extraction from plant material or chemical synthesis is possible. A sustainable alternative approach for menthol production could be a biotechnological-chemical two-step conversion, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low, if compared to the highly selective L3H enzymes from the biosynthetic pathway towards menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far, was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered further fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum . Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production and its additional ability to convert α-and β-pinene to verbenol and pinocarveol, respectively. Importance (−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi, which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.

Chemie Ingenieur TechnikVolume 92, Issue 9 p. 1235-1235 Poster Use of a methylotrophic organism for production of fine chemicals from methanol L. Pöschel, L. Pöschel DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorI. Marquardt, Corresponding Author I. Marquardt isabelle.marquardt@dechema.de DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanyCorrespondence: I. Marquardt (isabelle.marquardt@dechema.de), DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorM. Buchhaupt, M. Buchhaupt DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this author L. Pöschel, L. Pöschel DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorI. Marquardt, Corresponding Author I. Marquardt isabelle.marquardt@dechema.de DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanyCorrespondence: I. Marquardt (isabelle.marquardt@dechema.de), DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this authorM. Buchhaupt, M. Buchhaupt DECHEMA-Forschungsinstitut, Industrielle Biotechnologie, Theodor-Heuss-Allee 25, 60486 Frankfurt, GermanySearch for more papers by this author First published: 28 August 2020 https://doi.org/10.1002/cite.202055283AboutPDF 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. Volume92, Issue9Special Issue: 10. ProcessNet-Jahrestagung und 34. DECHEMA-Jahrestagung der Biotechnologen 2020: Processes for FutureSeptember 2020Pages 1235-1235 RelatedInformation

Monoterpenoids are widely used in industrial applications, e.g. as active ingredients in pharmaceuticals, in flavor and fragrance compositions, and in agriculture. Severe toxic effects are known for some monoterpenoids making them challenging compounds for biotechnological production processes. Some strains of the bacterium Pseudomonas putida show an inherent extraordinarily high tolerance towards solvents including monoterpenoids. An understanding of the underlying factors can help to create suitable strains for monoterpenoids de novo production or conversion. In addition, knowledge about tolerance mechanisms could allow a deeper insight into how bacteria can oppose monoterpenoid containing drugs, like tea tree oil. Within this work, the resistance mechanisms of P. putida GS1 were investigated using selected monoterpenoid-hypertolerant mutants. Most of the mutations were found in efflux pump promoter regions or associated transcription factors. Surprisingly, while for the tested monoterpenoid alcohols, ketone, and ether high efflux pump expression increased monoterpenoid tolerance, it reduced the tolerance against geranic acid. However, an increase of geranic acid tolerance could be gained by a mutation in an efflux pump component. It was also found that increased monoterpenoid tolerance can counteract efficient biotransformation ability, indicating the need for a fine-tuned and knowledge-based tolerance improvement for production strain development. Key points • Altered monoterpenoid tolerance mainly related to altered activity of efflux pumps. • Increased tolerance to geranic acid surprisingly caused by decreased export activity. • Reduction of export activity can be beneficial for biotechnological conversions.

Chemie Ingenieur TechnikVolume 92, Issue 9 p. 1233-1233 Poster High versatility of IPP methyltransferases enables synthesis of C6, C7, and C8 isoprenoid building blocks P. J. Haque, Corresponding Author P. J. Haque parab.haque@dechema.de DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanyCorrespondence: P. J. Haque (parab.haque@dechema.de), DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this authorM. Buchhaupt, M. Buchhaupt DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this author P. J. Haque, Corresponding Author P. J. Haque parab.haque@dechema.de DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanyCorrespondence: P. J. Haque (parab.haque@dechema.de), DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this authorM. Buchhaupt, M. Buchhaupt DECHEMA-Forschungsinstitut, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, GermanySearch for more papers by this author First published: 28 August 2020 https://doi.org/10.1002/cite.202055443AboutPDF 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. Volume92, Issue9Special Issue: 10. ProcessNet-Jahrestagung und 34. DECHEMA-Jahrestagung der Biotechnologen 2020: Processes for FutureSeptember 2020Pages 1233-1233 RelatedInformation

Due to their pleasant odor qualities and low odor thresholds, iso- and anteiso-fatty aldehydes represent promising candidates for applications in flavoring preparations. A novel cyanobacterial α dioxygenase from Crocosphaera subtropica was heterologously expressed in E. coli and applied for the biotechnological production of C11 - C15 branched-chain fatty aldehydes. The enzyme has a sequence identity of less than 40% to the well investigated α-dioxygenase from rice. Contrary to the latter, it efficiently transformed short chained fatty acids. The kinetic parameters of the α-dioxygenase towards unbranched and iso-branched-chain substrates were studied by means of an oxygen depletion assay. The transformation products (C12-C15 iso- and anteiso-aldehydes) were extensively characterized including their sensory properties. The aldehydes exhibited green-soapy, sweety odors with partial citrus-like, metallic, peppery, and savory-tallowy nuances. Moreover, the two C14 isomers showed particularly low odor threshold values of 0.2 and 0.3 ng/L air as determined by means of GC-olfactometry.

The "Bioflavour 2018-Biotechnology of Flavors, Fragrances, and Functional Ingredients" conference was held from September 18th to 21st, 2018 at the DECHEMA house in Frankfurt, Germany. The conference attracted more than 190 participants from over 25 countries, with about 40% share from industry. Particular sessions of Bioflavour 2018 focused on "flavor perception and biotechnology", "microbial cell factories", "novel pathways, enzymes, and biocatalysts", "technological and regulatory aspects of flavor and fragrance biotechnology", "advanced analytics and novel compounds", "plant biosynthesis and plant enzymes", "modern biotechnology in the world of wine", "receptors, flavors, and bioactives", and "bioprocess development and downstream processing". At Bioflavour 2018, both cutting-edge science from renowned academic research groups and current innovation from this modern biotechnology industry were presented and discussed. This special issue highlights a selection of 12 manuscripts from oral presentations and poster contributions.

Fatty aldehydes are among the most important flavor and fragrance compounds. Most biotechnological production approaches make use of the one step conversion of fatty acids from renewable sources by the enzymes α-dioxygenase (αDox) or carboxylic acid reductase (CAR). Their reaction mechanisms and cofactor dependencies are very different. In contrast to heme-containing αDox which requires only oxygen as cosubstrate, CAR needs NADPH and ATP, which is a clear argument for the application of a whole cell catalyst. Therefore we compared fatty acid biotransformations with growing Escherichia coli cells expressing αDox or CAR to investigate their suitability for fatty aldehyde and also fatty alcohol production. Our results show the main product of fatty acid conversions with αDox-expressing cells to be the expected Cn-1 aldehyde. However, 14% of the products consist of the corresponding alcohol, but in addition, 17% of the products consist of further shortened aldehydes, alcohols and acids that result from the consecutive activity of αDox and a putative endogenous fatty aldehyde dehydrogenase activity in E. coli. Conversely, CAR-expressing cells produced only the unshortened fatty aldehyde and alcohol, whereby the latter surprisingly accounts for at least 80% of the products. The considerably higher extend of aldehyde reduction of CAR-expressing cells was shown to be causally connected to the CAR-mediated fatty acid conversion. Our study provides an overview about the applicability of αDox- or CAR-based whole cell catalysts and gives a detailed description of side products as well as suggestions for tailored strain engineering.

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.

Chemie Ingenieur TechnikVolume 90, Issue 9 p. 1259-1259 VortragFree Access Deep eutectic solvents in biocatalysis S. Milker, Corresponding Author S. Milker sofia.milker@dechema.de DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanyCorrespondence: S. Milker (sofia.milker@dechema.de), DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., GermanySearch for more papers by this authorM. Hümmer, M. Hümmer DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this authorS. Kara, S. Kara Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestraße 15, 21073 Hamburg, GermanySearch for more papers by this authorA. Liese, A. Liese Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestraße 15, 21073 Hamburg, GermanySearch for more papers by this authorD. Holtmann, D. Holtmann DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this author S. Milker, Corresponding Author S. Milker sofia.milker@dechema.de DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanyCorrespondence: S. Milker (sofia.milker@dechema.de), DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., GermanySearch for more papers by this authorM. Hümmer, M. Hümmer DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this authorJ. Schrader, J. Schrader DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this authorS. Kara, S. Kara Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestraße 15, 21073 Hamburg, GermanySearch for more papers by this authorA. Liese, A. Liese Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestraße 15, 21073 Hamburg, GermanySearch for more papers by this authorD. Holtmann, D. Holtmann DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M, GermanySearch for more papers by this author First published: 24 August 2018 https://doi.org/10.1002/cite.201855279AboutPDF 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. References 1 Q. Zhang, K. De Oliveira Vigier, S. Royer, F. Jerome Chem. Soc. Rev. 2012, 41, 7108. Volume90, Issue9Special Issue: ProcessNet-Jahrestagung und 33. DECHEMA-Jahrestagung der Biotechnologen 2018September 2018Pages 1259-1259 ReferencesRelatedInformation

Deep eutectic solvents (DESs) based on (-)-menthol and fatty acids (octanoic, decanoic and dodecanoic acid) were investigated as reaction media for the lipase catalyzed esterification of the DES compounds itself to synthesize (-)-menthol fatty acid esters. The DES acts as reaction medium and substrate pool simultaneously without the need of adding any solvent. Candida rugosa lipase was active in the neat (-)-menthol:fatty acid DESs to synthesize (-)-menthol fatty acid esters. This example shows for the first time that a valuable product can be enzymatically produced using both components of a DES without any co-solvent. The addition of water to the DESs enhanced the reaction outcome likely due to interfacial activation of the enzyme. In biphasic reaction systems with an addition of 10 wt% of water to the DES phase, the conversion (7 d) of octanoic, decanoic and dodecanoic acid reached 50%, 83% and 71%, respectively. This corresponds to a batch productivity of 133 g L-1 d(-1) (24 h) and a final (-)-menthyl dodecanoate concentration of 957 mm (7 d) in the (-)-menthol:-dodecanoic acid DES. Closer investigation of this DES reaction system revealed that water addition and stirring speed are interacting parameters to optimize the process. The developed DES reaction systems represent neat reactant mixtures enabling the lipase catalyzed esterification under solvent-free conditions.

The structural diversity of terpenoids is limited by the isoprene rule which states that all primary terpene synthase products derive from methyl-branched building blocks with five carbon atoms. With this study we discover a broad spectrum of novel terpenoids with eleven carbon atoms as byproducts of bacterial 2-methylisoborneol or 2-methylenebornane synthases. Both enzymes use 2-methyl-GPP as substrate, which is synthesized from GPP by the action of a methyltransferase. We used E. coli strains that heterologously produce different C11-terpene synthases together with the GPP methyltransferase and the mevalonate pathway enzymes. With this de novo approach, 35 different C11-terpenes could be produced. In addition to eleven known compounds, it was possible to detect 24 novel C11-terpenes which have not yet been described as terpene synthase products. Four of them, 3,4-dimethylcumene, 2-methylborneol and the two diastereomers of 2-methylcitronellol could be identified. Furthermore, we showed that an E. coli strain expressing the GPP-methyltransferase can produce the C16-terpene 6-methylfarnesol which indicates the condensation of 2-methyl-GPP and IPP to 6-methyl-FPP by the E. coli FPP-synthase. Our study demonstrates the broad range of unusual terpenes accessible by expression of GPP-methyltransferases and C11-terpene synthases in E. coli and provides an extended mechanism for C11-terpene synthases.

The culture supernatant of Caldariomyces fumago strains grown in a minimal medium with fructose contains mainly the biotechnologically relevant enzyme chloroperoxidase (CPO) and only minor amounts of other proteins. Our approach to identify the nature of these proteins via peptide mass fingerprinting and transcriptome analysis demonstrated the presence of putative glycosyl hydrolase and glucose oxidase (GOx) enzymes. These activities had been described earlier as parts of the fungus´ halogenation machinery, as they provide CPO with the co-substrate H2O2. The GOx activity was found to have a pH optimum of 5. Compared to the wild type values, GOx activity and glucose-driven MCD chlorination activity in the culture of a white mutant were found to be strongly increased to values of 1–2 U mL−1. As most CPO-catalyzed peroxidation reactions also show pH optima at around 5, the C. fumago culture supernatant can provide a highly convenient CPO/GOx source for many reactions with in situ H2O2 production.

The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium Methylobacterium extorquens AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in M. extorquens AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in M. extorquens AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future.

Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL‐scale. In this paper, the application of a microtiter plate‐based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose‐like aroma compound 2‐phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms.

Streptomyces is a genus of Gram-positive bacteria that grow in various environments, and its shape resembles filamentous fungi. The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, and especially antibiotics. In submersed culture Streptomyces and other filamentous microorganisms can grow as free mycelium, pellets or intermediate growth forms. All growth forms can be beneficial for product formation depending on the microbial strain and the product of interest. Here, the effects of addition of particles on the growth and product formation of two Streptomyces strains were investigated. Streptomyces coelicolor was used as a model organism for the production of the antibiotic actinorhodin and Streptomyces avidinii was used to produce streptavidin. In all experiments, the addition of broken and porous SiO2 particles (120–200µm) to the culture medium led to an increased productivity. In order to evaluate the particle effect on actinorhodin production three different media with and without the addition of particles were compared. The productivity was strongly influenced by the choice of media and was further improved by the addition of particles. The enhancement factor between un-supplemented and supplemented cultures varied between 85% by using a complex medium and 160% in a minimal medium. The highest product concentration of 855mg actinorhodin L−1 was measured after 12 days in a complex medium by addition of 5g glass particles L−1. In contrast, the final concentration of streptavidin was not affected by the supplementation of glass beads (diameter: 0.25–0.5mm) to cultures of Streptomyces avidinii. Nevertheless, a kinetic effect occurred upon supplementation by particles, allowing an acceleration of product formation. The impact of the particle addition to the Streptomyces cultures on the sustainability of the bioprocess was proven by using the E-factor. The E-factor denotes the amount of waste generated per product equivalent. This evaluation shows the beneficial effects of the particle addition in both production systems. Due to the fact that the particle addition leads to a decreased E-factor a life cycle assessment should be performed to further evaluate the particle effect on the sustainability of the complete process chain.

Terpenoid flavor and fragrance compounds are of high interest to the aroma industry. Microbial production offers an alternative sustainable access to the desired terpenoids independent of natural sources. Genetically engineered microorganisms can be used to synthesize terpenoids from cheap and renewable resources. Due to its modular architecture, terpenoid biosynthesis is especially well suited for the microbial cell factory concept: a platform host engineered for a high flux toward the central C5 prenyl diphosphate precursors enables the production of a broad range of target terpenoids just by varying the pathway modules converting the C5 intermediates to the product of interest. In this review typical terpenoid flavor and fragrance compounds marketed or under development by biotech and aroma companies are given, and the specificities of the aroma market are discussed. The main part of this work focuses on key strategies and recent advances to engineer microbes to become efficient terpenoid producers.

Astaxanthin additions to animal diets predominantly serve as colorization aid to satisfy consumer expectations and desire for a consistent product with familiar coloration, e.g. the characteristic pink colorization of the flesh of species being produced by aquaculture. The heterobasidiomycetous yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous) can be used as natural feed source of astaxanthin. However, currently, the majority of astaxanthin used for the feed market is produced by chemical synthesis. We present a further step in direction of a competitive production of natural astaxanthin in an optimized bioprocess with non-genetically modified Phaffia rhodozyma. After medium optimization AXJ-20, a mutant strain of P. rhodozyma wild-type strain ATCC 96594, was able to grow to a cell dry weight concentration of over 114 g per kg of culture broth in a fed-batch process. In this bioprocess, where pH was lowered from 5.5 to 3.5 during the maturation phase, AXJ-20 produced the highest value reported for astaxanthin production with P. rhodozyma up to now: 0.7 g astaxanthin per kg of culture broth with a space-time-yield of 3.3 mg astaxanthin per kg of culture broth per hour. Lowering the pH during the bioprocess and increasing trace element and vitamin concentrations prevented loss of cell dry weight concentration in the maturation phase and proved to be critical for astaxanthin concentration and purity.

Microbial fuel cells (MFCs) are devices generating electrical current from a wide range of organic substrates by using bacterial metabolism. Integrations of MFCs into wastewater treatment plants seem to be the most likely application of this technology. Due to the fact that the current flow in a MFC is fundamentally produced by the metabolic activity of microorganisms, it would be desirable to elucidate the capacity of the microbial systems to optimize the energy extraction processes in MFCs. In this study, the correlation between the parameters X-BH (active heterotrophic biomass) and X-BA (active autotrophic biomass) from the established activated sludge model number 1 (ASM1) and the measured current flow in MFCs was investigated for the first time. The simulation protocol based on ASM1 shows a good congruence between measured and simulated effluent values for the wastewater treatment plant. Comparisons between the measured current densities and the simulated concentrations of active biomass showed linear correlations at substrate pulses and at different residence times of the substrate. Therefore, it can be concluded that the model parameter X-BH and X-BA of the ASM1 can be used to estimate the current output of a MFC in wastewater treatment plants. The identified correlations can be used to optimize operating conditions and to generate high current outputs of the MFCs based on simulations. (C) 2017 Elsevier Ltd. All rights reserved.

Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant.

Chemie Ingenieur TechnikVolume 88, Issue 9 p. 1337-1337 Poster Deep Eutectic Solvents als neuartige Reaktionsmedien in der Biokatalyse M. Hümmer, Corresponding Author M. Hümmer huemmer@dechema.de DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandDECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this authorProf. Dr. J. Schrader, Prof. Dr. J. Schrader DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this authorProf. Dr. A. Liese, Prof. Dr. A. Liese Institut für Technische Biokatalyse, TU Hamburg-Harburg, Denickestraße 15, 21073 Hamburg, DeutschlandSearch for more papers by this authorDr. D. Holtmann, Dr. D. Holtmann DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this author M. Hümmer, Corresponding Author M. Hümmer huemmer@dechema.de DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandDECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this authorProf. Dr. J. Schrader, Prof. Dr. J. Schrader DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this authorProf. Dr. A. Liese, Prof. Dr. A. Liese Institut für Technische Biokatalyse, TU Hamburg-Harburg, Denickestraße 15, 21073 Hamburg, DeutschlandSearch for more papers by this authorDr. D. Holtmann, Dr. D. Holtmann DECHEMA-Forschungsinstitut, Theodor-Heuss-Allee 25, 60486 Frankfurt a. M., DeutschlandSearch for more papers by this author First published: 29 August 2016 https://doi.org/10.1002/cite.201650343AboutPDF 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. Volume88, Issue9Special Issue: ProcessNet-Jahrestagung und 32. DECHEMA-Jahrestagung der Biotechnologen 2016September, 2016Pages 1337-1337 RelatedInformation