Downloaded from orbit.dtu.dk on: Oct 01, 2021 Lactobacilli and pediococci as versatile cell factories - Evaluation of strain properties and genetic tools Bosma, Elleke Fenna; Förster, Jochen; Nielsen, Alex Toftgaard Published in: Biotechnology Advances Link to article, DOI: 10.1016/j.biotechadv.2017.04.002 Publication date: 2017 Document Version Version created as part of publication process; publisher's layout; not normally made publicly available Link back to DTU Orbit Citation (APA): Bosma, E. F., Förster, J., & Nielsen, A. T. (2017). Lactobacilli and pediococci as versatile cell factories - Evaluation of strain properties and genetic tools. 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Biotechnology Advances xxx (xxxx) xxx–xxx Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Lactobacilli and pediococci as versatile cell factories – Evaluation of strain properties and genetic tools ⁎ Elleke F. Bosma, Jochen Forster, Alex Toftgaard Nielsen The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet B220, 2800 Kgs. Lyngby, Denmark ARTICLE INFO ABSTRACT Keywords: This review discusses opportunities and bottlenecks for cell factory development of Lactic Acid Bacteria (LAB), Strain development with an emphasis on lactobacilli and pediococci, their metabolism and genetic tools. In order to enable Genetic tools economically feasible bio-based production of chemicals and fuels in a biorefinery, the choice of product, Temperature substrate and production organism is important. Currently, the most frequently used production hosts include Stress tolerance Escherichia coli and Saccharomyces cerevisiae, but promising examples are available of alternative hosts such as Second generation biomass LAB. Particularly lactobacilli and pediococci can offer benefits such as thermotolerance, an extended substrate Third generation biomass range and increased tolerance to stresses such as low pH or high alcohol concentrations. This review will evaluate the properties and metabolism of these organisms, and provide an overview of their current biotechnological applications and metabolic engineering. We substantiate the review by including experimental results from screening various lactobacilli and pediococci for transformability, growth temperature range and ability to grow under biotechnologically relevant stress conditions. Since availability of efficient genetic engineering tools is a crucial prerequisite for industrial strain development, genetic tool development is extensively discussed. A range of genetic tools exist for Lactococcus lactis, but for other species of LAB like lactobacilli and pediococci such tools are less well developed. Whereas lactobacilli and pediococci have a long history of use in food and beverage fermentation, their use as platform organisms for production purposes is rather new. By harnessing their properties such as thermotolerance and stress resistance, and by using emerging high-throughput genetic tools, these organisms are very promising as versatile cell factories for biorefinery applications. 1. Introduction Lin and Xu, 2013; Mazzoli et al., 2014). This review provides an overview of the application of LAB for biotechnology purposes in the In order to satisfy the increasing world-wide demand for fuels and context of the biorefinery, with an emphasis on lactobacilli and chemicals in a sustainable way, alternatives to fossil resources are pediococci. First, the introduction will reflect on the requirements of needed. Whereas several renewable resources such as wind, water and substrates, organisms and products in a biorefinery. Second, this solar power can be used for supplying energy and fuels, microbial context will be used to examine the properties and metabolism of fermentation of biomass-derived sugars in a biorefinery is the most LAB. This will be illustrated with some experimental data on stress important alternative for chemical production. Some products such as tolerance, substrate utilization, products, temperature range, and lactic acid have a long history of commercial production via microbial genetic accessibility. Third, development of LAB into platform organ- fermentation, but for other products economically feasible production isms will be further explored by examining genetic tool development has not yet been achieved. The product, substrate as well as the and currently available examples of metabolic engineering towards production host all play a role in determining this. Currently, the most different end products. This section will provide an overview as well as frequently used production hosts include E. coli and S. cerevisiae, largely discussion of opportunities for further development. Lastly, concluding due to the availability of an array of efficient genetic engineering tools. remarks will be given on the suitability and future development However, these organisms are not always the most suitable hosts for possibilities of LAB and their products for biotechnology applications biochemicals production, and promising examples of alternative hosts in a biorefinery context. such as thermophiles or Lactic Acid Bacteria (LAB) are emerging due to a range of desirable features (Bosma et al., 2013; Gaspar et al., 2013; ⁎ Corresponding author. E-mail address: [email protected] (A.T. Nielsen). http://dx.doi.org/10.1016/j.biotechadv.2017.04.002 Received 13 February 2017; Received in revised form 29 March 2017; Accepted 3 April 2017 0734-9750/ © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). Please cite this article as: Bosma, E.F., Biotechnology Advances (2017), http://dx.doi.org/10.1016/j.biotechadv.2017.04.002 E.F. Bosma et al. Biotechnology Advances xxx (xxxx) xxx–xxx 1.1. Biorefinery considerations: product, substrate and production host produced at industrial scale, which is largely because of the high costs of the processes. Metabolic engineering has substantially broadened the Chemicals are typically divided into specialty, fine and bulk substrates that can be used by these organisms, ranging from xylose to chemicals. Biofuels and bulk chemicals such as plastics used for cellulose and alginate. However, there are many cases in which other packaging are low-cost and produced in large quantities. Specialty organisms have shown to be very promising alternative production and fine chemicals such as drugs and vitamins, on the other hand, are hosts with possible superior performance over model organisms. For more expensive and produced in smaller quantities (Budzianowski, example, organisms that are naturally and efficiently capable of using 2017). Most specialty, fine or bulk chemicals are today produced by complex substrates, are highly resistant to toxic compounds present in chemical synthesis, but in many cases, microbially produced chemicals or as substrate, medium, process or product, or are able to grow at high can directly replace existing products that are now produced from fossil temperatures (Bhalla et al., 2013; Boguta et al., 2014). Fermentation at resources. Furthermore, microorganisms can also be used to synthesize higher temperatures can improve process efficiency by reducing cooling compounds that are nowadays not produced via other ways, thereby costs and contamination risks and increasing product and substrate adding innovative products to the market (Budzianowski, 2017). solubility. Section 2.3.1 will provide more detailed examples of such Microbially produced chemicals that are direct replacements for exist- cases and explain the associated advantages and disadvantages. The ing chemicals need to have at least the same or lower price than the main downside of such non-model organisms is that they are less well- existing ones to be economically feasible. This is generally more studied and understood, and genetic tools are either absent, under- challenging for bulk than for specialty products, since bulk products developed or lack throughput, which limits their use as versatile are expected to remain very low-cost as long as oil prices do not platform organisms. Whereas it might be an option for selected dramatically increase. In order to enable the production of sustainable products to use a specific organism only for that single product, it is bulk biochemicals, it is therefore necessary to develop alternative and generally desirable in a biorefinery to use a single platform organism cost-efficient production methods. These considerations should be that can be metabolically engineered to produce any desired product in taken into account with the choice