Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production

Abstract

Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products.

Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand.

Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Fig. 1

Expression of n-butanol biosynthesis genes. a Scheme of a constructed n-butanol biosynthesis route in Synechocystis, with a competing PHB synthesis pathway deleted. Green arrows represent native genes, while blue, black and red arrows represent heterologously expressed genes. b Western blot of butanol biosynthesis enzymes from strain JA02 (phaJ ter adhE2 under P _psbA2) and JA04 (phaJ ter adhE2 under P _trc) at nitrogen replete (N+) and deplete (N−) conditions. 12 μg total protein was loaded from each sample

Fig. 2

Butanol production and growth curves at nitrogen replete (N+), nitrogen deplete (N−) and phosphorous deplete (P−) conditions. a Butanol accumulation after 14 days. Butanol production was abolished during nitrogen starvation if PHB synthase was present (JA01). Deletion of PHB synthase (JA02) resulted in increased butanol production during starvation. Enhanced expression of PhaJ, Ter and AdhE2 (JA04) increased butanol accumulation threefold. b Time course of butanol accumulation from strain JA02. Specific production of butanol increased up to twofold (P−) or threefold (N−). c Optical density in strain JA04 cultures. Cell densities remain constant during extended culturing in N− media. d Butanol titers and specific titers over time from strain JA04. The average specific productivities during day 4–14 of cultivation were 1.1 mg/gDCW/day (N+) and 2.7 mg/gDCW/day (N−). Error bars represent SD of biological triplicates

Fig. 3

Overview of metabolic effects on Synechocystis during nitrogen starvation. Metabolites (red squares) with potential influence on butanol synthesis were quantified at nitrogen replete (N+) and deplete (N−) conditions. Glycogen levels are relative to wild type at N−, which is defined as 100 %. Heterologous enzymes (in blue): xfpk (phosphoketolase, B. breve), phaJ (enoyl-CoA hydratase, A. caviae), ter (trans-enoyl-CoA reductase, T. denticola) and adhE2 (bifunctional aldehyde/alcohol dehydrogenase, C. acetobutylicum). Native enzymes (in green): phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), pta (phosphotransacetylase), ackA (acetate kinase), acs (acetyl-CoA synthetase). Error bars represent SD of biological duplicates for the acetyl-CoA, acetate and NAD⁺/NADH quantifications and biological triplicates for the glycogen and PHB quantifications. ND not detectable. Absolute metabolite values are listed in Additional file 1: Table S1

Fig. 4

Effect of phosphoketolase on flux balance analysis solutions in Synechocystis. a Flux through Xfpk in FBA solutions at autotrophic conditions when butanol production is used as objective function and biomass is fixed as a percentage of μmax. The photon uptake was constrained to 18.7 mmol/gDCW/h. b Phenotypic phase plane for butanol and biomass in autotrophic conditions. Presence of Xfpk increased theoretical butanol productivity

Fig. 5

Butanol production and growth rate of Xpfk strain at nitrogen replete (N+) and nitrogen deplete (N−) conditions. a Growth of butanol producing strains with (JA07) and without (JA04) Xfpk overexpression at nitrogen replete conditions. Panel: Specific growth rates calculated from slopes. b Time course of butanol accumulation. Phosphoketolase expression increased titer 1.7-fold at 8 days. c Butanol accumulation after 8 days. The specific production of butanol increased twofold at N+ when expressing an exogenous phosphoketolase (JA07). d Specific productivity of butanol over time. Phosphoketolase significantly increase specific productivity at one week of cultivation. Error bars represent SD of biological triplicates