Unprecedented Biomass and Fatty Acid Production by the Newly Discovered Cyanobacterium
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bioRxiv preprint doi: https://doi.org/10.1101/684944; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Unprecedented biomass and fatty acid production by the newly discovered cyanobacterium 2 Synechococcus sp. PCC 11901 3 Artur Włodarczyka, Tiago Toscano Selãoa, Birgitta Norlinga and Peter J. Nixona,b 4 5 aSchool of Biological Sciences, Nanyang Technological University, Singapore 6 bSir Ernst Chain Building- Wolfson Laboratories, Department of Life Sciences, Imperial College London, S. 7 Kensington Campus, London, SW7 2AZ, UK 8 9 10 Corresponding authors: Artur Włodarczyk, School of Biological Sciences, Nanyang Technological 11 University, Singapore. E-mail: [email protected] 12 Peter J. Nixon, Sir Ernst Chain Building- Wolfson Laboratories, Department of Life Sciences, Imperial 13 College London, UK. E-mail: [email protected] 14 15 16 17 18 19 20 21 22 23 24 bioRxiv preprint doi: https://doi.org/10.1101/684944; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 25 Abstract 26 Cyanobacteria, which use solar energy to convert carbon dioxide into biomass, are potential solar 27 biorefineries for the sustainable production of chemicals and biofuels. However, yields obtained with 28 current strains are still uncompetitive compared to existing heterotrophic production systems. Here we 29 report the discovery and characterization of a new cyanobacterial strain, Synechococcus sp. PCC 11901, 30 with potential for green biotechnology. It is transformable, has a short doubling time of ≈2 hours, grows 31 at high light intensities and a wide range of salinities and accumulates up to 18.3 g dry cell weight/L of 32 biomass – 2-3 times more than previously described for cyanobacteria - when grown in a modified 33 medium containing elevated nitrate, phosphate and iron. As a proof of principle, PCC 11901 engineered 34 to produce free fatty acids did so at unprecedented levels for cyanobacteria, with final yields reaching 35 over 6 mM (1.5 g/L), comparable to those achieved by heterotrophic organisms. 36 37 Abbreviations: 38 FFA – free fatty acids 39 OD730 – optical density measured at 730 nm wavelength 40 gDW – grams of dry weight 41 DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethylurea 42 RGB LED – red green blue light-emitting diode 43 44 Keywords: 45 Cyanobacteria, Synechococcus strain, free fatty acids, metabolic engineering, sustainable production 46 47 48 bioRxiv preprint doi: https://doi.org/10.1101/684944; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 49 1. Introduction. 50 The current production of commodity chemicals from fossil fuels results in the release of greenhouse 51 gases, such as CO2, into the atmosphere. Given concerns over the possible link between greenhouse 52 gases and climate change and the limited abundance of cheap fossil fuels1, alternative sustainable 53 approaches need to be developed to produce carbon-based chemicals on an industrial scale. Yeast and 54 bacteria are widely used biotechnology production platforms. However, their growth relies on the 55 addition of carbohydrates to the growth medium leading to a ’food vs. fuel’ dilemma, which will likely 56 drive the price of most carbon feedstocks up, fueled by the negative impact of global warming on food 57 crops2. Cyanobacteria have the potential to provide a completely sustainable solution3,4. These 58 evolutionary ancestors of algal and plant chloroplasts are gram-negative prokaryotic 59 oxyphotoautotrophs, able to convert carbon dioxide and inorganic sources of nitrogen, phosphorous 60 and microelements into biomass5. 61 Among cyanobacterial strains, the marine strain Synechococcus sp. PCC 70026 and several freshwater 62 strains, namely Synechocystis sp. PCC 68037, Synechococcus elongatus PCC 79428 and more recently 63 Synechococcus elongatus UTEX 29739, have become model organisms for both basic photosynthesis 64 research as well as the photoautotrophic production of different chemicals such as bioplastics10, biofuels 65 (ethanol11 and free fatty acids12,13) and specialized compounds like terpenoids14,15. However, the average 66 yields are often low compared to heterotrophic microbes, partly due to relatively slow growth and low 67 biomass accumulation. 68 In this work, we report the isolation and detailed characterization of the novel cyanobacterial strain 69 Synechococcus sp. PCC 11901 (hereafter PCC 11901) and demonstrate its potential for cyanobacterial 70 biotechnology. PCC 11901 tolerates temperatures up to a maximum of 43 °C, high light irradiances and 71 salinities, with average doubling times ranging from 2-3 hours in 1% CO2/air. We have also conducted a bioRxiv preprint doi: https://doi.org/10.1101/684944; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 72 parametric analysis to devise a new culture medium, termed MAD medium, which enables cultures of 73 PCC 11901 to produce the highest value of dry cell biomass of any cyanobacterial strain described so far. 74 Lastly, we tested the ability of PCC 11901 to efficiently convert CO2 into free fatty acids (FFA), a class of 75 commodity chemical used by a range of industries16. Although its current production from palm oil can 76 be considered as renewable17, growing world demand and extensive farming of palm oil trees in South 77 East Asia are causing irreversible deforestation of primordial rainforests and degradation of the natural 78 habitats in producing countries18. Yields of FFA obtained with PCC 11901 reached 6.16 mM (≈1.54 g/L) 79 after 7 days of cultivation, in the same range of productivity attained by heterotrophic hosts and 2.413 to 80 11.8-fold12 greater than yields previously achieved with cyanobacteria. 81 2. Results. 82 2.1. Strain isolation, identification and characterization. 83 As our primary goal was to isolate a fast-growing, preferably marine cyanobacterial strain that would 84 not compete for freshwater resources and could tolerate a wide range of abiotic stresses, we collected 85 samples at a local floating fish farm (an environment rich in waste nitrogen and phosphorous 86 compounds), located in the Johor river estuary in Singapore. Enrichment of water samples and 87 consecutive re-streaking on solid medium (see Materials and Methods) resulted in the isolation of a fast- 88 growing cyanobacterial colony. Inoculation into liquid AD7 medium lacking an organic carbon source, 89 which is used widely to grow marine cyanobacteria19,20 led to rapid growth, but only after a long lag 90 phase (≈24 hours), suggesting nutrient limitation. As most oceanic phytoplankton require cobalamin 21,22 91 (vitamin B12) for growth, and its concentrations in the seawater and oceans vary from 0 to 3 pM , we 92 supplemented cultures of the isolated xenic strain with 3 pM cobalamin, which alleviated the lag phase. 93 Upon dilution plating of the culture on solid medium with cobalamin, small transparent bacterial 94 colonies were found in the vicinity of the cyanobacterial colonies and both strains were further purified 95 by streaking on a 1:1 mixture of AD7 and LB medium. 16S rRNA sequencing coupled to phylogenetic bioRxiv preprint doi: https://doi.org/10.1101/684944; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 96 analyses revealed that the axenic cyanobacterial strain was a member of the Synechococcales group 97 (now deposited in the Pasteur Culture Collection as Synechococcus sp. PCC 11901) and that the closest 98 phylogenetic relatives to the companion heterotrophic bacterial strain belong to the marine 99 Thalassococcus genus23. Both the axenic and xenic strains of PCC 11901 grew equally well in the 100 presence of cobalamin, reaching an OD730 ≈23 after 72 hours (Fig. 1a). In contrast, growth of the axenic 101 strain was severely inhibited in the absence of added cobalamin with the observed residual growth 102 probably reflecting the retention of intracellular cobalamin or methionine reserves24 in the inoculum 103 (Fig. 1a). These results imply that PCC 11901 is auxotrophic, and that its growth depends on the 104 availability of cobalamin in the growth medium. 105 Although we did not sequence the genome of the isolated Thalassococcus strain, another 106 Thalassococcus sp. strain (SH-1) possesses cobalamin biosynthesis genes (GenBank accession number 107 CP027665.1). Given the previous evidence for the mutualistic interaction between cobalamin- 108 dependent microalgae and bacteria22, it is very likely that the Thalassococcus contaminant in the xenic 109 culture could serve as a natural symbiotic partner for the PCC 11901 strain providing it with cobalamin, 110 while consuming nutrients excreted by the cyanobacterium. 111 As in the case of PCC 700225, the PCC 11901 strain can grow mixotrophically in the presence of glycerol 112 and photoheterotrophically in the presence of glycerol and the herbicide DCMU, which inhibits 113 photosynthetic electron flow, although growth is impaired (Fig. S3c). Interestingly, this strain could not 114 utilize glucose for growth, as very little or no growth was observed after one week of incubation with 115 glucose and DCMU (Fig.