Photoautotrophic Production Research Articles

Tailoring regulatory components for metabolic engineering in cyanobacteria.

The looming climate crisis has prompted an ever-growing interest in cyanobacteria due to their potential as sustainable production platforms for the synthesis of energy carriers and value-added chemicals from CO2 and sunlight. Nonetheless, cyanobacteria are yet to compete with heterotrophic systems in terms of space-time yields and consequently production costs. One major drawback leading to the low production performance observed in cyanobacteria is the limited ability to utilize the full capacity of the photosynthetic apparatus and its associated systems, i.e. CO2 fixation and the directly connected metabolism. In this review, novel insights into various levels of metabolic regulation of cyanobacteria are discussed, including the potential of targeting these regulatory mechanisms to create a chassis with a phenotype favorable for photoautotrophic production. Compared to conventional metabolic engineering approaches, minor perturbations of regulatory mechanisms can have wide-ranging effects.

Photoautotrophic Production of Docosahexaenoic Acid- and Eicosapentaenoic Acid-Enriched Biomass by Co-Culturing Golden-Brown and Green Microalgae

Marine microalgae offer a sustainable alternative source for the human diet’s essential omega-3-fatty acids, including docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, none of them can produce DHA and EPA in a nutritionally balanced ratio of 1:1. As shown recently, the phototrophic co-cultivation of the golden-brown microalgae Tisochrysis lutea (DHA producer) with the green microalgae Microchloropsis salina (EPA producer) can provide microalgae biomass with a balanced DHA-to-EPA ratio with increased productivity compared to monocultures. This study evaluates whether other golden-brown (Isochrysis galbana) and green microalgae (Nannochloropsis oceanica, Microchloropsis gaditana) can enable the phototrophic batch production of omega-3 fatty acids in a nutritionally balanced ratio in co-culture. All co-cultivations applying a physically dynamic climate simulation of a repeated sunny summer day in Australia in LED-illuminated flat-plate gas lift photobioreactors resulted in increased biomass concentrations compared to their respective monocultures, achieving balanced DHA-to-EPA ratios of almost 1:1. Using urea instead of nitrate as a nitrogen source increased the EPA content by up to 80% in all co-cultures. Light spectra measurements on the light-adverted side of the photobioreactor showed that increased biomass concentrations in co-cultures could have been related to enhanced light use due to the utilization of different wavelengths of the two microalgae strains, especially with the use of green light (500–580 nm) primarily by golden-brown microalgae (I. galbana) and orange light (600–620 nm) predominantly used by green microalgae (N. oceanica). Phototrophic co-cultivation processes thus promise higher areal biomass yields if microalgae are combined with complimentary light-harvesting features.

Size-ftactioned photoautotrophic production in a shallow Bay.

CO2 assimilation measurements werecarried out in Concepción Bay (36"40'S, 73"0rW), toevalúatesizefractioned and total primary production in HC incubations. The P-I curve shows an assimilation nuinber of 3.82 mgC (mgChl-a)-1 h~1 and an a valué of 0.0076 (mgC(mgChl — a)'1 h~‘)/uE m-2 s-1, both parameters were used to estimate primary production in the water column using the Hermán & Platt model (1983). Integrated production was calculated as 0.97 gC m-2 d-1, four times higher than previous estimations reponed off the Chilean coast. At low irradiance (1= 109 uE m~2 s" ’). picoplankton and nanoplankton were responsible for 9.5% and 12.5% of the non-normalized production. whereas the net phytoplankton accounted for 64.6% of the photosynthetic assimilation.The chlorophyll normalized photosynthetic rate (productivity Índex) was higher for smaller than larger fractions, size dependant relationship with ecological significance at low light irradiances.

Characterization of Poly(3-hydroxybutyrate) (P3HB) from Alternative, Scalable (Waste) Feedstocks.

Bioplastics hold significant promise in replacing conventional plastic materials, linked to various serious issues such as fossil resource consumption, microplastic formation, non-degradability, and limited end-of-life options. Among bioplastics, polyhydroxyalkanoates (PHA) emerge as an intriguing class, with poly(3-hydroxybutyrate) (P3HB) being the most utilized. The extensive application of P3HB encounters a challenge due to its high production costs, prompting the investigation of sustainable alternatives, including the utilization of waste and new production routes involving CO2 and CH4. This study provides a valuable comparison of two P3HBs synthesized through distinct routes: one via cyanobacteria (Synechocystis sp. PCC 6714) for photoautotrophic production and the other via methanotrophic bacteria (Methylocystis sp. GB 25) for chemoautotrophic growth. This research evaluates the thermal and mechanical properties, including the aging effect over 21 days, demonstrating that both P3HBs are comparable, exhibiting physical properties similar to standard P3HBs. The results highlight the promising potential of P3HBs obtained through alternative routes as biomaterials, thereby contributing to the transition toward more sustainable alternatives to fossil polymers.

Fluctuating pH for efficient photomixotrophic succinate production

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO2 fixation rates and CO2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0 g L−1 after 10 days under shake flask conditions. These results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

Chemolithoautotrophic denitrification intensifies nitrogen loss in the Eastern Arabian Sea Shelf waters during sulphidic events

The Eastern Arabian Sea Shelf i.e. Western Indian Continental Shelf (WICS) – a known biogeochemical hotspot is characterized by monsoonal upwelling, seasonal O2 deficiency, extremely high N2O build-up and sulphidic events. The frequency and duration of the sulphidic events have increased over the last two decades, but their impact on the pelagic N cycling, N budget, and N2O dynamics is poorly constrained. Thus, to address these problems and assess their implications on WICS biogeochemistry, we carried out physico-chemical measurements, 15N-labeled incubations and bag incubations on five transects over the shelf during the sulphidic event (September-October) of 2011. We observed very high rates of sulphide-driven chemolithotrophic denitrification (1885–5825 nM N2 d−1) in the sulphidic, nitrate-depleted waters, and its potential occurrence in the sulphide-free, nitrate-replete waters (460–3137 nM N2 d−1), along with high transient N2O production, and comparably low rates of anammox (0–119 nM N2 d−1) and DNRA (0–45 nM N d−1). Despite the predominant cloud cover during the monsoon season, we could for the first time show the satellite image of a large colloidal sulphur (S0) plume associated with the sulphidic event off Western India providing further evidence of extensive sulphide oxidation coupled to denitrification. Sulphide-driven denitrification (mean rate = 2697 nM N2 d−1) appeared to be the dominant N loss process during the anoxic regime (September-October) replacing the chemoorganotrophic (i.e. heterotrophic) denitrification (342 nM N2 d−1) that predominates during the preceding suboxic regime (July-August). Overall, the highest sulphide-driven denitrification rate over the WICS was found to be the second highest among the anoxic coastal systems of the world. Furthermore, simultaneous consumption of NOx− and S2− at a ratio close to the theoretical value in the anaerobic incubations of chemocline waters indicated that the sulphide-driven denitrifiers were fixing carbon. The estimated dark C production (0.21 g C m−2 d−1) due to chemolithoautotrophic denitrification was 18% of the photoautotrophic production and accounted for 15% of the total column productivity. Based on our conservative estimates, the chemolithoautotrophic denitrification was responsible for the removal of 0.4 Tg of fixed N and 0.57 Tg of sulphide, and fixation of 0.1 Tg of carbon annually in the shelf waters. Thus, the sulphidic event impacted the biogeochemistry and ecology, and modulated the N loss pathways and rates over the WICS. With the expansion and intensification of OMZs induced by global climate change, and the spreading of dead zones due to increasing anthropogenic activities, chemolithoautotrophic denitrification is likely to become increasingly significant in oceanic N cycle and impact the N budgets of shallow marine systems particularly.

Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli

BackgroundWhole-cell biotransformation is a promising emerging technology for the production of chemicals. When using heterotrophic organisms such as E. coli and yeast as biocatalysts, the dependence on organic carbon source impairs the sustainability and economic viability of the process. As a promising alternative, photosynthetic cyanobacteria with low nutrient requirements and versatile metabolism, could offer a sustainable platform for the heterologous production of organic compounds directly from sunlight and CO2. This strategy has been applied for the photoautotrophic production of sucrose by a genetically engineered cyanobacterium, Synechocystis sp. PCC 6803 strain S02. As the key concept in the current work, this can be further used to generate organic carbon compounds for different heterotrophic applications, including for the whole-cell biotransformation by yeast and bacteria.ResultsEntrapment of Synechocystis S02 cells in Ca2+-cross-linked alginate hydrogel beads improves the specific sucrose productivity by 86% compared to suspension cultures during 7 days of cultivation under salt stress. The process was further prolonged by periodically changing the medium in the vials for up to 17 days of efficient production, giving the final sucrose yield slightly above 3000 mg l−1. We successfully demonstrated that the medium enriched with photosynthetically produced sucrose by immobilized Synechocystis S02 cells supports the biotransformation of cyclohexanone to ε-caprolactone by the E. coli WΔcscR Inv:Parvi strain engineered to (i) utilize low concentrations of sucrose and (ii) perform biotransformation of cyclohexanone to ε-caprolactone.ConclusionWe conclude that cell entrapment in Ca2+-alginate beads is an effective method to prolong sucrose production by the engineered cyanobacteria, while allowing efficient separation of the cells from the medium. This advantage opens up novel possibilities to create advanced autotroph–heterotroph coupled cultivation systems for solar-driven production of chemicals via biotransformation, as demonstrated in this work by utilizing the photosynthetically produced sucrose to drive the conversion of cyclohexanone to ε-caprolactone by engineered E. coli.

Simultaneous photoautotrophic production of DHA and EPA by Tisochrysis lutea and Microchloropsis salina in co-culture

Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day–night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mgDHA gCDW−1, and 23 mgEPA gCDW−1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures.Graphical

Comparative assessment of environmental impacts of 1st generation (corn feedstock) and 3rd generation (carbon dioxide feedstock) PHA production pathways using life cycle assessment

Polyhydroxyalkanoates (PHA) are bio-based and biodegradable alternatives to conventional plastic types and have the potential to reduce the environmental impacts along the life cycle. In comparison to already established production routes for PHA (heterotrophic production) based on renewable feedstock like glucose (first generation feedstock), novel production routes, such as the photoautotrophic production of PHA based on CO2 as feedstock (third generation feedstock) could offer new perspectives with regard to the reduction in the environmental impacts. To quantify the environmental impacts of PHA produced via photoautotrophic and heterotrophic production pathways, life cycle assessment (LCA) methodology based on ISO 14040/44 was applied, thus conducting a first of its kind comparative study for PHA based on third generation feedstock. The results show that the photoautotrophic production of PHA has advantages in comparison to heterotrophic PHA based on glucose originating from corn as feedstock in all the assessed environmental impact categories, thus showing the environmental potential of novel production routes for bioplastics. Additionally, the results of the LCA show that the chloroform-based extraction method, commonly used in the downstream processes of both the technologies, has a significant contribution of environmental impacts in the production of PHA. Therefore, the reduction of chloroform loss during the extraction process can reduce its environmental impact. Our results indicate that PHA production from CO2 using the photoautotrophic production route is a promising technology with regard to the environmental impacts when compared to the heterotrophic production based on glucose feedstock.

A scalable model for EPA and fatty acid production by Phaeodactylum tricornutum.

Large-scale photoautotrophic production of microalgae has the potential to provide a sustainable supply of omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) for human and animal nutrition. This study presents a kinetic model for the EPA-producing microalga Phaeodactylum tricornutum in photoautotrophic conditions, with light and nitrogen being the growth limiting factors. The model was developed using a dataset obtained from bench-scale (5 L) cultures and was successfully validated against pilot-scale (50 L) cultures. This model is the first to predict the biomass and total fatty acid accumulation along with the EPA concentrations in the biomass and total fatty acid fraction for microalgae. The model was used to develop an optimized repeated-batch strategy; implementation of this led to increases in the biomass and EPA productivities of 50 and 20% respectively. This clearly indicates the potential of the model to be used as a tool in the design, optimization and scale-up of microalgal systems for EPA production.

Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.

Developing Rhodobacter sphaeroides for cathodic biopolymer production

In this work, Rhodobacter sphaeroides was identified as a potential cathodic production strain for photoautotrophic production processes. First, a stable cultivation in a bioelectrochemical system (BES) was established under conditions in which hydrogen produced by a poised cathode served as an electron donor. It was shown that both the introduction of a plasmid vector and exposure to the corresponding antibiotic selection pressure caused a strong improvement in both cathodic biofilm formation and electrochemical properties. A quantitative proteomic analysis identified key players in the molecular adaptation to biofilm growth on the cathodic surface. Furthermore, biofilm formation kinetics were quantified by optical coherence tomography measurements, which showed a strong tendency for biofilm formation together with a robust biofilm architecture. A media switch to N2-limited conditions resulted in increased cathodic poly(3-hydroxybutyrate) (PHB) accumulation, suggesting R. sphaeroides as a potential strain for photoautotrophic PHB production in future industrial applications.

Photoautotrophic production of renewable ethylene by engineered cyanobacteria: Steering the cell metabolism towards biotechnological use.

Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethylene now used for commercial applications is produced exclusively from nonrenewable petroleum sources, while competitive biotechnological production systems do not yet exist. This review focuses on the currently developed photoautotrophic bioproduction strategies that enable direct solar-driven conversion of CO2 into ethylene, based on the use of genetically engineered photosynthetic cyanobacteria expressing heterologous ethylene forming enzyme (EFE) from Pseudomonas syringae. The emphasis is on the different engineering strategies to express EFE and to direct the cellular carbon flux towards the primary metabolite 2-oxoglutarate, highlighting associated metabolic constraints, and technical considerations on cultivation strategies and conditional parameters. While the research field has progressed towards more robust strains with better production profiles, and deeper understanding of the associated metabolic limitations, it is clear that there is room for significant improvement to reach industrial relevance. At the same time, existing information and the development of synthetic biology tools for engineering cyanobacteria open new possibilities for improving the prospects for the sustainable production of renewable ethylene.

Production of hydroxy fatty acids and its effects on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

Microalgal lipids and fatty acids are important components for achieving biofuel because of their potential high productivity. Although fatty acids that have a hydroxy group adjacent to the end of the acyl chain might be an important chemical feedstock, most algae do not accumulate it. To produce (ω−1)-hydroxy fatty acids from 3-hydroxybutylyl-CoA, an intermediate for polyhydroxybutylate biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, we expressed a gene for the promiscuous 3-ketoacyl-ACP synthase III from Alicyclobacillus acidocalderius (aaKASIII) by the cpc560 promoter. To supply 3-hydroxybutyryl-CoAs for aaKASIII, the phaC gene for polyhydroxybutylate polymerase was deleted, and the phaAB genes for 3-hydroxybutyryl-CoA synthesis from Cupriavidus necator were overexpressed. The genetically modified strain synthesized 15-hydroxyhexadecanoic acid, 17-hydroxyoctadecanoic acid, and 17-hydroxyoctadec-9-enoic acid, and accumulated approximately 2.1 mol% of (ω−1)-hydroxy fatty acids in total fatty acids under illumination with 70 μmol photons m−2 s−1, although its growth was severely retarded. Under weak light (35 μmol photons m−2 s−1) conditions, the strain grew as well as the wild-type and showed lower hydroxy fatty acids (0.04 mol%) accumulation than that at higher illumination levels. The photosynthetic activity of this strain was lower than that of wild-type cells, suggesting that high light conditions enhanced hydroxy fatty acids production and inhibited photosynthesis. (ω−1)-Hydroxy fatty acids were not predominantly observed in the galactolipids from thin-layer chromatography, which are the major lipid classes in cyanobacteria. To the best of our knowledge, this is the first report on photoautotrophic production of fatty acids possessing a functional group near the end of the acyl chain in cyanobacteria.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Mapping the performance of photobioreactors for microalgae cultivation. Part II: equatorial and tropical climate zone

AbstractBACKGROUNDConsidering that the greatest proportion of microalgae commercial production is realized outdoors, it is critically important to highlight the role of the local climate in the performance of the processes. Currently, there is a tendency to identify locations with suitable climates for the large‐scale implementation of microalgae‐based processes. Therefore, this study proposed to map the performance of photobioreactors for microalgae culture. Fourteen types of climates distributed in 33 geographical locations representative of the equatorial and tropical zones of the world were evaluated. The simulated climatic elements were solar irradiation, temperature and photoperiod.RESULTSThe results showed that the tropical with dry winter (Aw), tropical with dry summer (As), tropical monsoon (Am), tropical fully humid (Af), hot steppe (BSh) and hot desert (BWh) climates favored the system productivities. The highest biomass productivity (0.347 g L–1 d–1) and lipid (0.048 g L–1 d–1) were registered in the Aw simulation.CONCLUSIONThe results of this study provide a useful database of the effects of climate for the microalgae photoautotrophic production. © 2020 Society of Chemical Industry

Directed Reaction Engineering Boosts Succinate Formation of Synechocystis sp. PCC 6803_Δsll1625.

It is known that Synechocystis sp. PCC 6803 carrying a partial deletion of the succinate dehydrogenase (Synechocystis_∆sll1625) secretes succinate during aerobic cultivation with continuous illumination and in the presence of CO2 . Maximal succinate titers of 2mM (236 mg L-1 ) are reported. CO2 is identified as a crucial parameter for product formation, however, a detailed characterization of different cultivation conditions is still missing. Here the focus is on further reaction engineering to improve the photoautotrophic production of succinate using Synechocystis_∆sll1625. Therefore the impact of light availability, illumination regimes, nutrient availability, and external pH on product formation are investigated. Results obtained in this study reveal the importance of these parameters on the formation of succinate and cultivation with light/dark cycles increases the succinate concentration to 3mM (354mgL-1 ) after 28 days of cultivation. Furthermore, cultivation in unbuffered medium under ambient CO2 conditions even doubled the final succinate titer to 4mM (472mgL-1 ) after 28 days. Taking biomass concentrations into account, a maximal yield of succinate on biomass of 215mgSucc gCDW -1 is achieved, which is the highest so far reported for the production of succinate utilizing Synechocystis as host organism.

Photosynthetic Co-Production of Succinate and Ethylene in A Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801.

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of PcpcB, the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.

Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid

BackgroundCyanobacteria, a group of photosynthetic prokaryotes, are being increasingly explored for direct conversion of carbon dioxide to useful chemicals. However, efforts to engineer these photoautotrophs have resulted in low product titers. This may be ascribed to the bottlenecks in metabolic pathways, which need to be identified for rational engineering. We engineered the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801 to produce succinate, an important platform chemical. Previously, engineering of the model cyanobacterium S. elongatus PCC 7942 has resulted in succinate titer of 0.43 g l−1 in 8 days.ResultsBuilding on the previous report, expression of α-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and phosphoenolpyruvate carboxylase yielded a succinate titer of 0.6 g l−1 in 5 days suggesting that PCC 11801 is better suited as host for production. Profiling of the engineered strains for 57 intermediate metabolites, a number of enzymes and qualitative analysis of key transcripts revealed potential flux control points. Based on this, we evaluated the effects of overexpression of sedoheptulose-1,7-bisphosphatase, citrate synthase and succinate transporters and knockout of succinate dehydrogenase and glycogen synthase A. The final construct with seven genes overexpressed and two genes knocked out resulted in photoautotrophic production of 0.93 g l−1 succinate in 5 days.ConclusionWhile the fast-growing strain PCC 11801 yielded a much higher titer than the model strain, the efficient photoautotrophy of this novel isolate needs to be harnessed further for the production of desired chemicals. Engineered strains of S. elongatus PCC 11801 showed dramatic alterations in the levels of several metabolites suggesting far reaching effects of pathway engineering. Attempts to overexpress enzymes deemed to be flux controlling led to the emergence of other potential rate-limiting steps. Thus, this process of debottlenecking of the pathway needs to be repeated several times to obtain a significantly superior succinate titer.

Overexpression of bicarbonate transporters in the marine cyanobacterium Synechococcus sp. PCC 7002 increases growth rate and glycogen accumulation

BackgroundSynechococcus sp. PCC 7002 is an attractive organism as a feedstock and for photoautotrophic production of biofuels and biochemicals due to its fast growth and ability to grow in marine/brackish medium. Previous studies suggest that the growth of this organism is limited by the HCO3− transport across the cytoplasmic membrane. Tools for genetic engineering are well established for this cyanobacterium, which makes it possible to overexpress genes of interest.ResultsIn this work, we overexpressed two different native Na+-dependent carbon transporters viz., SbtA and BicA in Synechococcus sp. PCC 7002 cells under the influence of a strong light-inducible promoter and a strong RBS sequence. The overexpression of these transporters enhanced biomass by about 50%, increased intracellular glycogen about 50%, and increased extracellular carbohydrate up to threefold. Importantly, the biomass and glycogen productivity of the transformants with air bubbling was even higher than that of WT cells with 1% CO2 bubbling. The overexpression of these transporters was associated with an increased carotenoid content without altering the chl a content.ConclusionsOur work shows the utility of increased carbon transport in improving the growth as well as product formation in a marine cyanobacterium and will serve to increase the utility of this organism as a potential cell factory.