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Team:Stony Brook

Interlab Results

Stony Brook iGEM 2018

Project Description: Engineering Synechococcus elongatus PCC 7942 to Export Sucrose for Carbon Sequestration and Use as an Industrial Feedstock

Introduction:

PLA plastic, ethanol biofuel, and various industrial compounds use sucrose as a feedstock. Sucrose from corn drives up food prices for developing nations and uses unsustainable amounts of fertilizers and fossil fuels in its production [1]. Sucrose from sugarcane leads to deforestation and disruption of the natural ecosystems in the Amazon rainforest [2].

Photosynthetic cyanobacteria strains such as Synechococcus elongatus PCC 7942 can produce sucrose more efficiently than both corn and sugarcane. S. elongatus fixes CO2 and secretes roughly 80% of fixed carbon as sucrose without the need for cell-harvesting [3]. Crops such as corn and sugarcane only allocate 20% of their fixed carbon as sucrose, and large amounts of land and fertilizer are required for their production. Because of the high efficiencies involved, sucrose production from cyanobacteria may be an effective method for carbon sequestration by converting CO2 into sugars that can be used to make stable, high-value products such as bio-plastics.

Ducat et al. and Xuefeng et al. both successfully engineered S. elongatus to produce and secrete sucrose in large quantities; however, the process has not been commercialized because of harvesting costs and inefficient photobioreactor design [4].

Our project intends to make cyanobacteria carbon sequestration industrially viable. We will characterize sucrose-related genes and native promoters in S. elongatus under the Biobrick standard. We will construct novel gene circuits, which make sucrose production and secretion easy to toggle. We will also make open-source protocols for culturing and using directed evolution with our strain of cyanobacteria.

Sucrose Biobricks:

S. elongatus naturally produces sucrose in saltwater to balance osmotic pressure [5]. We intend to modify S. elongatus to produce and export sucrose under freshwater conditions in order to reduce harvesting costs associated with salt-removal. We aim to construct two BioBricks: one which produces sucrose, and one which exports it.

The protein responsible for the rate-determining step in S. elongatus is sucrose phosphate synthase (Sps), but it requires sodium ions to function [6]. We will instead codon optimize the sps gene from another cyanobacteria strain, Synechocystis sp. PCC 6803, which shows sucrose production in the absence of NaCl [6].

The sucrose permease protein (CscB), derived from E. coli, can pump sucrose out of S. elongatus [3]. There is currently a BioBrick for cscB, but it does not meet RFC10 standards due to illegal cut-sites. We aim to codon optimize cscB for our strain and make it BioBrick compatible.

In order to initially characterize the sps and cscB BioBricks, we will clone them in Dr. Susan Golden’s IPTG-inducible vector pAM2991. We will use a sucrose/d-glucose assay to compare the effects of Sps and CscB on extracellular and intracellular sucrose concentrations against the wild type.

Promoter Biobricks:

S. elongatus enters stationary phase upon producing and secreting sucrose because carbon is redirected from growth [3]. Because this phenotype is unfavorable and easily selected out of our cyanobacteria, we want to induce sucrose production and secretion once S. elongatus has reached maximum cell density. Researchers such as Ducat et al. have used IPTG-inducible promoters; however, such promoters are leaky, and using IPTG is not cost-effective or easy to toggle in an industrial setting [3]. Additionally, despite S. elongatus being a model photosynthetic bacterium, there is a lack of characterized promoters in the registry.

We intend to characterize a variety of promoters in S. elongatus as BioBricks. These promoters will be valuable tools for the rest of the synthetic biology community. We will characterize the ferric- and ferrous-ion repressible promoters associated with the idiA and isiAB genes [7,8], the high-light inducible promoter of psbA2 [9], and strong constitutive promoters of cpcB native to S. elongatus and Synechocystis sp. PCC 6803 [10]. We will use Dr. Golden’s promoterless luxAB vector pAM1414 to compare their relative strengths.

Application:

After characterizing the promoters as BioBricks, we intend to use them to induce sucrose production and secretion by combining them with our cscB and sps BioBricks. Using some of the aforementioned promoters in an industrial setting has numerous advantages. The iron-repressible promoters initiate transcription with high on/off ratios once iron concentrations fall below millimolar thresholds [7,8]. Since iron is a micronutrient in our cyanobacteria’s BG-11 media, this is easily tunable. We can adjust the concentration of the media such that iron is completely exhausted upon reaching maximum cell density, which would trigger sucrose production. Alternatively, the psbA2 promoter would make sucrose production easy to toggle. It is only expressed when S. elongatus is exposed to ¼ peak sunlight intensity or greater (roughly 500 µmoles photons/s/m2) [9]. We can grow S. elongatus under low light until it reaches stationary phase. Upon achieving maximum cell density, we can increase light levels, triggering sucrose production. Using this promoter also keeps sucrose production in phase with light intensity, which is desirable as sucrose acts as an electron acceptor and should mitigate oxidative stress under intense light.

Lab Automation and Open-Source Protocols:

Complementing our main project, we will provide detailed, open-source protocols for culturing cyanobacteria as a contribution to the synthetic biology community. We specifically want to automate protocols that use UV-based random mutagenesis and selection for different phenotypes such as high growth-rate and high sucrose production, as a proof of concept for directed evolution of our strain.

This year, our team had the honor of winning an Opentrons OT-2 robot that we will use for lab automation. We will create and add protocols specifically for cyanobacteria to the Opentrons repository. Additionally, we plan on incorporating our own hardware modules (such as a simple, adjustable LED panel) into the OT-2 to facilitate working with cyanobacteria. With the OT-2, we will also assess the impact of various factors—light intensity, bicarbonate concentration, temperature, and light penetration—on growth rate and integrate this data into our protocols.

Directed Evolution:

Because directed evolution is a powerful yet time-consuming tool, we will design automated protocols for the OT-2, which will select for desired phenotypes. Using a Raspberry Pi camera and custom well-plates, we can easily measure optical density and select for fast-growing strains. In conjunction with a sucrose assay, we can select for higher yield as well.

Summary:

By making detailed, open-source protocols and characterizing novel promoter and protein-coding BioBricks, our project has the potential to accelerate the use of cyanobacteria in industrial synthetic biology and carbon sequestration.

References:

1. Pimentel, D. (2003). Ethanol Fuels: Energy Balance, Economics, and Environmental Impacts Are Negative. Natural Resources Research
2. Jusys, T. (2017). A confirmation of the indirect impact of sugarcane on deforestation in the Amazon. Journal of Land Use Science
3. Ducat DC, Avelar-Rivas JA, Way JC, Silver PA (2012). Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol.
4. Qiao, C., Duan, Y., Zhang, M., Hagemann, M., Luo, Q. and Lu, X. (2017). Effects of lowered and enhanced glycogen pools on salt-induced sucrose production in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. Appl Environ Microbiol.
5. Klahn S, Hagemann M. (2011). Compatible solute biosynthesis in cyanobacteria. Environ. Microbiol.
6. B.W. Abramson, B. Kachel, D.M. Kramer, D.C. Ducat. (2016). Increased photochemical efficiency in cyanobacteria via an engineered sucrose sink. Plant Cell Physiol.
7. Michel KP, Pistorius EK, Golden SS. (2001). Unusual regulatory elements for iron deficiency induction of the idiA gene of Synechococcus elongatus PCC 7942. J Bacteriol
8. Kunert A., Vinnemeier J., Erdmann N., Hagemann M. (2003). Repression by Fur is not the main mechanism controlling the iron-inducible isiAB operon in the cyanobacterium Synechocystis sp. PCC 6803. FEMS Microbiol.
9. Tsinoremus, N, Schaefer, M, and Golden, S. (1994). Blue and red light reversibly control psbA expression in the cyanobacterium Synechococcus sp. strain PCC 7942. J. Biol. Chem.
10. Zhou J, Zhang H, Meng H, Zhu Y, Bao G, Zhang Y, Li Y, Ma Y. (2014). Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Sci Rep

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