Abstract
The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.
Highlights
BATCH CULTURE PRODUCTIVITY Nitrogen replete and nitrogen deplete batch cultures of WT, SA01, ∆glgC, and SA13 were analyzed over 48 h for chlorophyll a, Dry cell weight (DCW), fatty acid methyl esters (FAMEs), Reducing carbohydrates (RCs), and Organic acids (OAs)
Chlorophyll a and dry cell weight Bulk biomass accumulation in nutrient replete batch cultures yielded an increase in chlorophyll a content of 12- to 15-fold over 48 h (Figure S1A in Supplementary Material), and DCW accumulated 4- to 5-fold (Figure S1C in Supplementary Material)
In nitrogen-deplete cultures, growth attenuation was suggested by unchanging chlorophyll a content and DCWs that were within the range of error relative to inoculum DCWs over the time course (Figures S1B,D in Supplementary Material)
Summary
Photosynthetic metabolism generates a wide range of biomolecules fundamental to energy, agriculture, and health (Durrett et al, 2008; Hu et al, 2008; Atsumi et al, 2009; Lubner et al, 2009; Lindberg et al, 2010; Lu, 2010; Niederholtmeyer et al, 2010; Kilian et al, 2011; Wahlen et al, 2011; Ducat et al, 2012; Elliott et al, 2012; Work et al, 2013; Sorek et al, 2014). Efficient energy conversion, and metabolic adaptability, photosynthetic microorganisms (PSMs) including genetically tractable unicellular algae and cyanobacteria have received substantial attention for synthesizing biofuel precursors via native or transgenic processes (Ferrari et al, 1971; Cascon and Gilbert, 2000; Heifetz, 2000; Lee, 2001; Schmer et al, 2008; Rodolfi et al, 2009; Elliott et al, 2011; Fore et al, 2011; Liu et al, 2011; Radakovits et al, 2011; Soratana and Landis, 2011; Rosgaard et al, 2012; Bentley et al, 2013; Gronenberg et al, 2013; Leite et al, 2013; Möllers et al, 2014; Davies et al, 2015). Microbial C12 synthesis has been achieved via transgenics in both heterotrophic and photoautotrophic hosts (Ohlrogge et al, 1995; Lu et al, 2008; Liu et al, 2011; Radakovits et al, 2011; Lennen and Pfleger, 2012), offering diverse opportunities in production platforms
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