8) Engineering microbial cell factories for new directions in biotechnology

Microbes have been used for centuries as cell factories to produce valuable resources for human consumption. In many cases, naturally occurring mixed microbial communities can be exploited for that purpose. One can however also make artificial communities by mixing microbes that do not work together naturally, but make an excellent team in the lab to produce valuable compounds. One step further is to produce microbes that do not naturally occur by genetically modifying them in the lab so that they perform the required task. This session will focus on these topics with three speakers that are experts in these research fields.


David R. Johnson, Eawag:
Indiviual microbial genotypes typically do nog effectively perform a wide range of metabolic processes. Instead, different microbial genotypes speecialize at performing only specific sets of metabolic process. Why is hit? What prevents one mirobial genotype from effectively performing a wide range of metabolic processes? We present a general theory for why different microbial genotypes specialize at performing only specific sets of metabolic processes. The central hypothesis is that different metabolic processes are in biochemical conflict with each other, thus causing them to segregate into different microbial genotypes over evolutionary time. We next provide experimental evidence supporting the main predictions of the theory using a synthetic denitrifying microbial community as a model system. Finally, we propose how information about biochemical conflicts between different metabolic processes could enable the rational design of microbial communities to achieve engineering objectives. Namely, we present a framework for predicting how best to distribute different metabolic processes across different microbial genotypes to maximize the performance of a desired metabolic transformation.

Jinyoung Jung, Yeungnam University:
The key operating parameters from improving the nitrogen removal rate (NRR) in a sequencing batch reactor (SBR) for deammonification were investigated. Denitrification with the addition of an organic source was beneficial for improving the NRR from 0.50.01 kg N m-3 d-1 to 0.530.01 kg N m-3 d-1 by removing the nitrate produced as a by-product of ANAMMOX. Unlike the gradual increase of the specific activity for AOB, the specific ANAMMOX activity (SAA) was maximized when an ammonium concentration supplied after sub-feeding phase was increased from 20 to 100 mg L-1, which increased the NRR from 0.530.01 kg N m-3 d-1 to 0.790.01 kg N m-3 d-1. In the whole experimental period, the granule the granule size smaller than 100 ㎛ accounted for 52.50.9%, making the largest contribution to the activity for AOB and denitrifiers. However, the granule size larger than 100㎛ made the greatest contribution (83.80.5%) to SAA. The feasibility of using the derivate of pH and OPR as indirect parameters to control the NRR was verified.

Diana Z. Sousa, Wageningen University & Research:
Syngas can be produced from carbon-containing material and is a suitable feedstock for biotechnological processes. Anaerobes can utilize syngas, but the natural products of this conversion are generally limited to acetate and ethanol. At our research group, we aim at broadening the variety of products that can be produced from syngas by developing tailored anaerobic microbial consortia. We established a co-culture of Clostridium autoethanogenum, an acetogen that converts syngas to acetate and ethanol, together with Clostridium kluyveri, a bacterium able to perform chain elongation. This resulted in the production of a mixture of C4 and C6 acids and alcohols from syngas. Using a transcriptomics and studying the co-culture behavior in a chemostat-system we have attempted to unravel its functioning. Results indicate that production of hydrogen by C. kluyveri stimulates the metabolism of C. autoethanogenum, resulting in more ethanol formation. This has a subsequent effect on the production levels of C4 and C6 products by C. kluyveri. This type of interactions is important for product optimization and tuning of current and future synthetic co-culture designs.

Richard van Kranenburg, Corbion & Laboratory of Microbiology Wageningen University:
Thermophilic bacilli are gaining increasing attention for their potential as bacterial cell factories to support the biobeased economy. Their ability to efficiently use lignocellulosic sugars make them particularly suited for fermentation of biomass substrates. In our laboratory we isolated various genetically accessible strains from compost. For Bacillus smithii we focussed on genetic tool development for metabolic engineering purposes. After establishing the standard tools for genome engineering by homologous recombination, we expanded our repertoire with CRISPR-Cas9 application. For this, we developed a protocol to apply mesophilic Streptococcus pyogenes Cas9 for genome editing, an approach that seems applicable to many more thermophiles. In addition, we isolated, characterized and applied thermostable Cas9. Our work underlines the potential of thermophilic bacilli as chassis organisms supported by the rapid development of genetic tools.

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