Corynebacterium glutamicum as a platform strain for the production of a broad variety of terpenoids

Corynebacterium glutamicum is a natural carotenoid producing bacterium used in the million-ton-scale amino acid biotechnology that has been engineered for isoprenoid production1. The native membrane-bound carotenoid decaprenoxanthin is a rare C50 carotenoid. Volatile terpenoids such as valencene2 and patchoulol3 could be produced upon deletion of the first step of the specific carotenoid pathway and heterologous expression of the FPP synthase gene ispA from E. coli and terpene synthases from plant origin. However, these strains produced a yet unidentified carotenoid and only when all carotenoid biosynthetic genes were deleted, a colorless strain resulted. Expressing a codon optimized ADS from Artemisia annua in the white strain, amorphadiene, the volatile precursor for artemisinin was produced. For production of volatile terpenoids a dodecane overlay was used, a condition in which C. glutamicum benefits from its robust myco-membrane. Recently, we showed production of membrane-bound carotenoids with different length and/or cyclization status: bicyclic C50 sarcinaxanthin4, bicyclic C40 astaxanthin5, the linear lycopene6 and the linear C50 bisanhydrobacterioruberin7. This indicated that the C. glutamicum myco-membrane accepts these linear and bicyclic carotenoids. Please click Additional Files below to see the full abstract

Design-of-Experiment-Guided Establishment of a Fermentative Bioprocess for Biomass-Bound Astaxanthin with <i>Corynebacterium glutamicum</i>

Corynebacterium glutamicum is prominent in the industrial production of secreted amino acids. Notably, it naturally accumulates the carotenoid pigment decaprenoxanthin in its membranes. Metabolic engineering enabled the production of astaxanthin. Here, a bioprocess for astaxanthin production in lab-scale stirred bioreactors was established by a DoE-guided approach to optimize the basic process parameters pH, rDOS, aeration rate as well as inoculation cell density. The DoE-guided approach to characterize 2 L scale cultivation revealed that the pH showed the strongest effect on the product formation. Subsequently, an optimum at pH 8, an aeration rate of 0.25 vvm, 30% rDOS and an initial optical density of 1 was established that allowed production of 7.6 ± 0.6 mg L−1 astaxanthin in batch mode. These process conditions were successfully transferred to a fed-batch process resulting in a high cell density cultivation with up to 60 g CDW L−1 biomass and 64 mg L−1 astaxanthin and thus demonstrating an about 9-fold improvement compared to optimal batch conditions. Moreover, pH-shift experiments indicate that the cells can quickly adapt to a change from pH 6 to 8 and start producing astaxanthin, showing the possibility of biphasic bioprocesses for astaxanthin production

Modeling and Simulating the Aerobic Carbon Metabolism of a Green Microalga Using Petri Nets and New Concepts of VANESA

In this work we present new concepts of VANESA, a tool for modeling and simulation in systems biology. We provide a convenient way to handle mathematical expressions and take physical units into account. Simulation and result management has been improved, and syntax and consistency checks, based on physical units, reduce modeling errors. As a proof of concept, essential components of the aerobic carbon metabolism of the green microalga Chlamydomonas reinhardtii are modeled and simulated. The modeling process is based on xHPN Petri net formalism and simulation is performed with OpenModelica, a powerful environment and compiler for Modelica. VANESA, as well as OpenModelica, is open source, free-of-charge for non-commercial use, and is available at: http://agbi.techfak.uni-bielefeld.de/vanesa

Modeling and Simulating the Aerobic Carbon Metabolism of a Green Microalga Using Petri Nets and New Concepts of VANESA

In this work we present new concepts of VANESA, a tool for modeling and simulation in systems biology. We provide a convenient way to handle mathematical expressions and take physical units into account. Simulation and result management has been improved, and syntax and consistency checks, based on physical units, reduce modeling errors. As a proof of concept, essential components of the aerobic carbon metabolism of the green microalga Chlamydomonas reinhardtii are modeled and simulated. The modeling process is based on xHPN Petri net formalism and simulation is performed with OpenModelica, a powerful environment and compiler for Modelica. VANESA, as well as OpenModelica, is open source, free-of-charge for non-commercial use, and is available at: http://agbi.techfak.uni-bielefeld.de/vanesa

From Aquaculture to Aquaculture: Production of the Fish Feed Additive Astaxanthin by <i>Corynebacterium glutamicum</i> Using Aquaculture Sidestream

Circular economy holds great potential to minimize the use of finite resources, and reduce waste formation by the creation of closed-loop systems. This also pertains to the utilization of sidestreams in large-scale biotechnological processes. A flexible feedstock concept has been established for the industrially relevant Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin. In this study, we aimed to use a preprocessed aquaculture sidestream for production of carotenoids, including the fish feed ingredient astaxanthin by C. glutamicum. The addition of a preprocessed aquaculture sidestream to the culture medium did not inhibit growth, obviated the need for addition of several components of the mineral salt’s medium, and notably enhanced production of astaxanthin by an engineered C. glutamicum producer strain. Improved astaxanthin production was scaled to 2 L bioreactor fermentations. This strategy to improve astaxanthin production was shown to be transferable to production of several native and non-native carotenoids. Thus, this study provides a proof-of-principle for improving carotenoid production by C. glutamicum upon supplementation of a preprocessed aquaculture sidestream. Moreover, in the case of astaxanthin production it may be a potential component of a circular economy in aquaculture

Patchoulol Production with Metabolically Engineered Corynebacterium glutamicum

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L&minus;1 and volumetric productivities of up to 18 mg L&minus;1 d&minus;1