Letter Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX
pubs.acs.org/synthbio
High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria Ryan L. Clark,† Gina C. Gordon,†,‡ Nathaniel R. Bennett,† Haoxiang Lyu,† Thatcher W. Root,† and Brian F. Pfleger*,† †
Department of Chemical and Biological Engineering, University of Wisconsin − Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, United States ‡ Microbiology Doctoral Training Program, University of Wisconsin − Madison, 1550 Linden Drive, Madison, Wisconsin 53706, United States S Supporting Information *
ABSTRACT: As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for Llactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU. KEYWORDS: cyanobacteria, biocontainment, CO2-concentrating mechanism, carboxysome, horizontal gene transfer, natural competence
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metabolite.13,14 Auxotrophies to synthetic metabolites eliminate this escape mechanism, but current methods require supplementation with nonstandard amino acids10,11 or nucleic acids12 that are not readily available or cost-effective for largescale cultivation of phototrophs. Due to the low abundance of CO2 in aquatic systems, cyanobacteria and eukaryotic algae have evolved CO2concentrating mechanisms (CCM) to increase the local concentration of CO2 near the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) enzyme inside of cellular protein microcompartments called carboxysomes.15 Disrupting this mechanism in cyanobacteria through deletion of carbxysome shell proteins16 or inorganic carbon transporters17 imposes a high-CO2 concentration requirement (HCR) for growth. HCR caused by CCM disruption has been suggested as a containment mechanism for photoautotrophic microorganisms because an industrial cultivation environment could be supplemented with waste CO2, while the relatively CO2-
any researchers have genetically engineered cyanobacteria for photosynthetic production of fuels and commodity chemicals directly from CO2 and sunlight.1−3 Systems level analysis has begun to assess the viability of socalled “photosynthetic biorefinery” processes for their feasibility to create replacements for chemicals sourced from nonrenewable feedstocks.4 Strategies for using photoautotrophs (plants, cyanobacteria, or algae) to produce fuels or chemicals in large scale require cultivation across large surface areas, greatly increasing the risk of contaminating the surrounding natural ecosystems with genetically modified or non-native species. Therefore, biocontainment strategies are needed to alleviate risk and reduce fear in the general public regarding use of genetically modified organisms. To date, most biocontainment research has focused on the use of toxin and antitoxin pairs, kill switches,5−8 or the establishment of auxotrophies to natural9 or synthetic10−12 metabolites. Despite increasingly robust circuits, kill switch mechanisms are susceptible to deactivation through genetic drift and require the development of sensing systems to trigger the switch.7,13 Auxotrophies to natural metabolites can be circumvented through natural sources of the required © XXXX American Chemical Society
Received: October 16, 2017 Published: January 10, 2018 A
DOI: 10.1021/acssynbio.7b00377 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology depleted natural environment would be unable to support the growth of CCM-lacking photoautotrophs.18,19 While modeling efforts support the concept, little work has been done to experimentally validate this containment strategy. Horizontal gene transfer has played a major role in the evolution of cyanobacteria, allowing beneficial genetic changes to spread between organisms.20 The model cyanobacterial species Synechococcus sp. strain PCC7002 (PCC7002),21 Synechococcus elongatus sp. PCC7942,22 and Synechocystis sp. PCC680323 are particularly amenable to horizontal gene transfer due to their high natural competence, a characteristic which is frequently leveraged for genetic engineering. Any containment mechanism developed for use with one of these highly competent organisms must consider the potential of escape through horizontal gene transfer. Therefore, strategies for reducing genetic escape must be simultaneously developed. Here, we experimentally validate a HCR-containment strategy using the model cyanobacterium PCC7002 in which the CCM was disrupted, resulting in a high-CO2 requirement for growth. Because this containment mechanism involves only gene deletion, it is robust to the issues of genetic drift encountered by kill switch mechanisms. There are no proximal environments containing CO2 concentrations sufficient for growth of CCM-lacking cyanobacteria, so environmental supplementation is not a possible escape mechanism. One potentially likely escape mechanism is through horizontal gene transfer of the highly conserved CCM-related genes.24 Therefore, we investigated the risk of escape through horizontal gene transfer and mitigated the risk through the deletion of natural competence-related genes. The dual containment mechanism was implemented in a previously characterized strain of PCC7002 engineered to produce L-lactate and we observed no negative impact on growth or L-lactate production.
Figure 1. Growth of Wild Type PCC7002 (circles) and PCC7002 ΔccmK2K1LMN (triangles) at varying PCO2. Data is given as the average specific growth rate of three replicates (error bars smaller than the data symbols). Values in parentheses are the best fit parameters of the model in eq 1 and are represented by the solid curves.
gible growth after 72 h in ambient air, but grew at the same rate as Wild Type PCC7002 in 5% CO2 (0.05 atm CO2) or greater, verifying the expected high-CO2 requirement for 7002_RLC01. Unsurprisingly, μmax did not differ by much between Wild Type PCC7002 and PCC7002 ΔccmK2K1LMN as RuBisCO saturation should result in similar growth rates assuming similar RuBisCO content. It is interesting to note that the previously measured KCO2 for RuBisCO in PCC7002 was 185 μM CO2, the concentration of a aqueous phase in equilibrium with a gas phase containing 0.09% CO2 (9 × 10−3 atm CO2),28 a value almost double the K C O 2 measured for PCC7002 ΔccmK2K1LMN (0.05% CO2, 5 × 10−3 atm CO2, Figure 1). The kinetics of CO2 fixation in CCM-lacking mutants is thought to be controlled by RuBisCO kinetics as the cytoplasmic CO2 concentration is equal to the media CO2 concentration.18 This discrepancy could be due to differences in temperature between the in vitro RuBisCO kinetic assay (25 °C) and the in vivo experiments in this work (38 °C). To test the effectiveness of the HCR containment mechanism, we grew cultures of Wild Type PCC7002 and PCC7002 ΔccmK2K1LMN in 10% CO2 to an OD730 of approximately 3 and then plated cells on nonselective Media A in ambient air or 5% CO2. For Wild Type PCC7002 an equivalent number of colonies were recovered in both conditions. For PCC7002 ΔccmK2K1LMN, 5 × 108 CFU mL−1 were recovered in 5% CO2, but no colonies were observed in ambient air where 4 mL of culture (2 × 109 cells) were plated. The survival rate of PCC7002 ΔccmK2K1LMN in ambient air was therefore less than 5 × 10−10 CFU−1 (Table 1), 2 orders of magnitude lower than the NIH recommended limit of one escape event in 108 CFU.14 While the HCR containment mechanism was sufficiently effective, escape could occur through horizontal gene transfer in
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RESULTS AND DISCUSSION To investigate the survival of CCM-lacking cyanobacteria under differing CO2 concentrations, we deleted the ccmK2K1LMN operon from PCC7002 using a kanamycin resistance marker as described previously.25 This operon encodes the proteins CcmK2 and CcmK1, which make up the faces of the carboxysome shell; CcmL, which forms the vertices of the carboxysome shell; CcmM, which enables aggregation of RuBisCO; and CcmN, which facilitates encapsulation of the aggregated RuBisCO by anchoring the carboxysome shell proteins to the surface of the aggregate.26 The resulting strain, PCC7002 ΔccmK2K1LMN (7002_RLC01), as well as wild type PCC7002 were grown in culture tubes bubbled with various concentrations of CO2. The exponential growth rate of each culture was measured at cell densities low enough for light attenuation to be minimal (OD730 < 0.1).27 The resulting exponential growth data as a function of CO2 concentration were fit to a Monod growth rate model (eq 1). μ(PCO2) =
μmax PCO2 K CO2 + PCO2
(1)
Here, μ is the specific growth rate, PCO2 is the partial pressure of CO2 in the gas phase, μmax is the maximum specific growth rate, and KCO2 is the half-maximum growth rate constant for CO2. The exponential growth data and Monod model fits are shown in Figure 1 for Wild Type PCC7002 and PCC7002 ΔccmK2K1LMN. PCC7002 ΔccmK2K1LMN exhibited negli-
Table 1. Colony Forming Units per mL for Culture Plated in 5% CO2 or Ambient Air
B
plating condition
5% CO2
ambient air
Wild Type PCC7002 PCC7002 ΔccmK2K1LMN
5 × 108 5 × 108
5 × 108
