The microbial food revolution

Our current food system relies on unsustainable practices, which often fail to provide healthy diets to a growing population. Therefore, there is an urgent demand for new sustainable nutrition sources and processes. Microorganisms have gained attention as a new food source solution, due to their low carbon footprint, low reliance on land, water and seasonal variations coupled with a favourable nutritional profile. Furthermore, with the emergence and use of new tools, specifically in synthetic biology, the uses of microorganisms have expanded showing great potential to fulfil many of our dietary needs. In this review, we look at the different applications of microorganisms in food, and examine the history, state-of-the-art and potential to disrupt current foods systems. We cover both the use of microbes to produce whole foods out of their biomass and as cell factories to make highly functional and nutritional ingredients. The technical, economical, and societal limitations are also discussed together with the current and future perspectives

Recombinant β-carotene production by yarrowia lipolytica - assessing the potential of micro-scale fermentation analysis in cell factory design and bioreaction optimization

The production of β-carotene has become increasingly interesting within the biotechnological industry due to a rising demand for safer and more natural colorants, nutritional supplements, and antioxidants. A recent study has described the potential of Yarrowia lipolytica as a β-carotene-producing cell factory, reporting the highest titer of recombinant β-carotene produced to date. Finding the best conditions to maximize production and scaling up the process to full scale, a costly and time-consuming process, it is often a bottleneck in biotechnology. In this work, we explored the benefits of using micro-fermentation equipment to significantly reduce the time spent on design and optimization of bioreaction conditions, especially in the early stages of process development. In this proof-of-concept study, a β-carotene producing Y. lipolytica strain was tested in micro-fermentations partly to assess the robustness of the cell factory design and partly to perform media optimization. The medium optimization led us to an improvement of up to 50% in the yield of β-carotene production in the best of the conditions. Overall, the micro-fermentation system had a high degree of reliability in all tests

Unraveling the potential of non-conventional yeasts in biotechnology.

Cost-effective microbial conversion processes of renewable feedstock into biofuels and biochemicals are of utmost importance for the establishment of a robust bioeconomy. Conventional baker's yeast Saccharomyces cerevisiae, widely employed in biotechnology for decades, lacks many of the desired traits for such bioprocesses like utilization of complex carbon sources or low tolerance towards challenging conditions. Many non-conventional yeasts (NCY) present these capabilities, and they are therefore forecasted to play key roles in future biotechnological production processes. For successful implementation of NCY in biotechnology, several challenges including generation of alternative carbon sources, development of tailored NCY and optimization of the fermentation conditions are crucial for maximizing bioproduct yields and titers. Addressing these challenges requires a multidisciplinary approach that is facilitated through the 'YEAST4BIO' COST action. YEAST4BIO fosters integrative investigations aimed at filling knowledge gaps and excelling research and innovation, which can improve biotechnological conversion processes from renewable resources to mitigate climate change and boost transition towards a circular bioeconomy. In this perspective, the main challenges and research efforts within YEAST4BIO are discussed, highlighting the importance of collaboration and knowledge exchange for progression in this research field

Synthetic biology tools to engineer microbial communities for biotechnology

Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial consortia for diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and the development of new tools to construct synthetic consortia and program their behaviors, will vastly expand the functions that can be performed by communities of interacting microorganisms. Here, we review recent advancements in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss ongoing and emerging efforts to apply consortia for various biotechnological applications, and suggest future applications

Synthetic biology tools for engineering Yarrowia lipolytica

The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools

Microbial chassis development for natural product biosynthesis

Engineering microbial cells to efficiently synthesize high-value-added natural products has received increasing attention in recent years. In this review, we describe the pipeline to build chassis cells for natural product production. First, we discuss recently developed genome mining strategies for identifying and designing biosynthetic modules and compare the characteristics of different host microbes. Then, we summarize state-of-the-art systems metabolic engineering tools for reconstructing and fine-tuning biosynthetic pathways and transport mechanisms. Finally, we discuss the future prospects of building next-generation chassis cells for the production of natural products. This review provides theoretical guidance for the rational design and construction of microbial strains to produce natural products

Metabolic engineering of clostridium cellulovorans to improve butanol production by consolidated bioprocessing.

Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (ter), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica.

Background: Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches. Results: To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed. Conclusions: Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend