A Critical Review of Genome Editing and Synthetic Biology Applications in Metabolic Engineering of Microalgae and Cyanobacteria

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi‐omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value‐added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.     

聚球藻7942光自养培养的碳代谢和能量代谢

代谢通量分析是研究微藻光自养培养过程中CO2和光能利用的一个非常有用的工具。本研究建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。研究结果表明,CO2固定是代谢能量和还原力消耗的主要途径,分别约占总消耗能量的85%和70%。研究还发现在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80g/molATP和2.95g/molATP,但基于总吸收光能的细胞得率和对应的光能转换效率则随着光强的增加而降低。     

Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production

Cyanobacteria are ideal metabolic engineering platforms for carbon-neutral biotechnology because they directly convert CO₂ to a range of valuable products. In this study, we present a computational assessment of biochemical production in Synechococcus sp. PCC 7002 (Synechococcus 7002), a fast growing cyanobacterium whose genome has been sequenced, and for which genetic modification methods have been developed. We evaluated the maximum theoretical yields (mol product per mol CO₂ or mol photon) of producing various chemicals under photoautotrophic and dark conditions using a genome-scale metabolic model of Synechococcus 7002. We found that the yields were lower under dark conditions, compared to photoautotrophic conditions, due to the limited amount of energy and reductant generated from glycogen. We also examined the effects of photon and CO₂ limitations on chemical production under photoautotrophic conditions. In addition, using various computational methods such as minimization of metabolic adjustment (MOMA), relative metabolic change (RELATCH), and OptORF, we identified gene-knockout mutants that are predicted to improve chemical production under photoautotrophic and/or dark anoxic conditions. These computational results are useful for metabolic engineering of cyanobacteria to synthesize value-added products.     

Flux balance analysis of Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions

Quantification of carbon flux distribution in the metabolic network of microalgae remains important to understand the complex interplay between energy metabolism, carbon fixation, and assimilation pathways. This is even more relevant with respect to cyclic metabolism of microalgae under light–dark cycle. In the present study, flux balance analysis (FBA) was carried out for an indigenous isolate Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions. A shift in intracellular flux distribution was predicted during transition from nutrient sufficient phase to nutrient starvation phase of growth. Further, dynamic flux analysis (dFBA) was carried out to capture light–dark metabolism over discretized pseudo steady state time intervals. Our key findings include the following: (i) unlike heterotrophic condition, oxidative pentose phosphate (PP) pathway, and Krebs cycle were relatively inactive under photoautotrophic growth; (ii) in both growth conditions, while transhydrogenation reaction was highly active, glyoxalate shunt was found to be nonoperative; (iii) flux distribution during transition period was marked with up regulation of carbon flux toward nongrowth associated (NGA) maintenance energy, oxidative phosphorylation, and photophosphorylation; (iv) redirection of carbon flux from polysaccharide and neutral lipid resulted in up regulation of Krebs cycle flux in the dark phase; (v) elevated glycolytic and acetyl-CoA flux were coupled with induction of neutral lipid during light cycle of the growth; (vi) significantly active photophosphorylation in the light phase was able to satisfy cellular energy requirement without need of oxidative PP pathway; and (vi) unlike static FBA, dFBA predicted an unaltered NGA maintenance energy of 1.5 mmol g^?1 DCW h^?1.     

Characterization of energy conversion of Synechococcus sp. PCC7942 under photoautotrophic conditions based on metabolic flux and chlorophyll fluorescence analysis

Energy conversion efficiency of photoautotrophic microalgae plays an important role in the utilization of light energy for cell growth and production of metabolites. To understand the utilization of light energy, Synechococcus sp. PCC7942 was cultivated at different incident light intensities of 15.8, 47.3, and 94.6 μmol/m²/sec in continuous culture. The influence of light on the carbon and energy metabolism of microalgae was investigated by combining metabolic flux analysis (MFA) and chlorophyll fluorescence analysis (CFA). Results showed that the yields of biomass based on ATP (Y _ATP) and total light energy (Y _E) both declined with increasing light, and the maximal values of Y _ATP and Y _E were estimated to be 4.73 g/mol-ATP, and 17.10 × 10^?3 g/kJ respectively, at the examined conditions. The overall efficiency of energy conversion against total absorbed energy changed with the varying irradiances. However, the actual conversion efficiency of total energy based on CFA was almost constant, regardless of the different irradiances used in the present study.     

Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions

Chlorella pyrenoidosa was cultivated under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. The influence of light on the carbon and energy metabolism of microalgae was investigated by the use of metabolic flux analysis. The respiratory activity of microalgae in the light was assessed from the autotrophic flux distribution. Results showed that the glycolytic pathway, tricarboxylic acid cycle and mitochondrial oxidative phosphorylation maintained high activities during illumination, indicating little effect of light on these pathways, while the flux through the pentose phosphate pathway during illumination was very small due to the light-mediated regulation. The theoretical yields of biomass on ATP decreased in the following order: heterotrophic culture>mixotrophic culture>autotrophic culture, and a significant amount of the available ATP was required for maintenance processes in microalgal cells. The energy conversion efficiency between the supplied energy to culture, the absorbed energy by cells and the free energy conserved in ATP were analyzed for the different cultures. Analysis showed that the heterotrophic culture generated more ATP from the supplied energy than the autotrophic and mixotrophic cultures. The maximum thermodynamic efficiency of ATP production from the absorbed energy, which was calculated from the metabolic fluxes at zero growth rate, was the highest in the heterotrophic culture and as low as 16% in the autotrophic culture. By evaluating the energy economy through the energy utilization efficiency, it was found that the biomass yield on the supplied energy was the lowest in the autotrophic cultivation, and the cyclic culture gave the most efficient utilization of energy for biomass production.     

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.     

2,3 Butanediol production in an obligate photoautotrophic cyanobacterium in dark conditions via diverse sugar consumption

Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO₂ into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12 h:12 h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0 g L⁻¹ in 10 d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using cyanobacteria as industrial chemical-producing microbes.     

Flux balance analysis of photoautotrophic metabolism

Photosynthesis is the principal process responsible for fixation of inorganic carbon dioxide into organic molecules with sunlight as the energy source. Potentially, many chemicals could be inexpensively produced by photosynthetic organisms. Mathematical modeling of photoautotrophic metabolism is therefore important to evaluate maximum theoretical product yields and to deeply understand the interactions between biochemical energy, carbon fixation, and assimilation pathways. Flux balance analysis based on linear programming is applied to photoautotrophic metabolism. The stoichiometric network of a model photosynthetic prokaryote, Synechocystis sp. PCC 6803, has been reconstructed from genomic data and biochemical literature and coupled with a model of the photophosphorylation processes. Flux map topologies for the hetero-, auto-, and mixotrophic modes of metabolism under conditions of optimal growth were determined and compared. The roles of important metabolic reactions such as the glyoxylate shunt and the transhydrogenase reaction were analyzed. We also theoretically evaluated the effect of gene deletions or additions on biomass yield and metabolic flux distributions.     

Regulating the ratio of photoautotrophic to heterotrophic metabolic activities in photoheterotrophic culture of Euglena gracilis and its application to α-tocopherol production

Methods of regulating the ratio of photoautotrophic to heterotrophic growth rates in photoheterotrophic culture of Euglena gracilis were investigated. In normal photoheterotrophic culture (in the presence of excess organic carbon), the cells grew mainly by organic carbon assimilation (heterotrophic metabolism). The relative contribution of photoautotrophic metabolism increased with the increase in the light supply coefficient, the increase in the CO₂ concentration in the aeration gas and the decrease in the feed rate of organic carbon source. However, limiting the organic carbon supply was the most effective method of shifting the metabolic balance to the photoautotrophic side. In the presence of excess organic carbon source, the -tocopherol contents of the cells in photoheterotrophic culture were low even when the light supply coefficient and CO₂ concentration in the aeration gas were high. By limiting the organic carbon supply to the photoheterotrophic culture, the intracellular content of -tocopherol increased to the same level as those obtained in photoautotrophic cultures.     

Progress and perspective on cyanobacterial glycogen metabolism engineering

As important oxygenic photoautotrophs, cyanobacteria are also generally considered as one of the most promising microbial chassis for photosynthetic biomanufacturing. Diverse synthetic biology and metabolic engineering approaches have been developed to enable the efficient harnessing of carbon and energy flow toward the synthesis of desired metabolites in cyanobacterial cell factories. Glycogen metabolism works as the most important natural carbon sink mechanism and reserve carbon source, storing a large portion of carbon and energy from the Calvin-Benson-Bassham (CBB) cycle, and thus is traditionally recognized as a promising engineering target to optimize the efficacy of cyanobacterial cell factories. Multiple strategies and approaches have been designed and adopted to engineer glycogen metabolism in cyanobacteria, leading to the successful regulation of glycogen synthesis and storage contents in cyanobacteria cells. However, disturbed glycogen metabolism results in weakened cellular physiological functionalities, thereby diminishing the robustness of metabolism. In addition, the effects of glycogen removal as a metabolic engineering strategy to enhance photosynthetic biosynthesis are still controversial. This review focuses on the efforts and effects of glycogen metabolism engineering on the physiology and metabolism of cyanobacterial chassis strains and cell factories. The perspectives and prospects provided herein are expected to inspire novel strategies and tools to achieve ideal control over carbon and energy flow for biomanufacturing.     

Heterotrophic cultivation of microalgae for pigment production: A review

Pigments (mainly carotenoids) are important nutraceuticals known for their potent anti-oxidant activities and have been used extensively as high end health supplements. Microalgae are the most promising sources of natural carotenoids and are devoid of the toxic effects associated with synthetic derivatives. Compared to photoautotrophic cultivation, heterotrophic cultivation of microalgae in well-controlled bioreactors for pigments production has attracted much attention for commercial applications due to overcoming the difficulties associated with the supply of CO₂ and light, as well as avoiding the contamination problems and land requirements in open autotrophic culture systems. In this review, the heterotrophic metabolic potential of microalgae and their uses in pigment production are comprehensively described. Strategies to enhance pigment production under heterotrophic conditions are critically discussed and the challenges faced in heterotrophic pigment production with possible alternative solutions are presented.     

Metabolic flux network and analysis of fermentative hydrogen production

Fermentative hydrogen production (FHP) has received a great R & D interest in recent decades, as it offers a potential means of producing H₂ from a variety of renewable resources, even wastewater via a low energy continuous process. Various extracellular metabolites including ethanol, acetate, butyrate and lactate can be produced during the fermentation, building a complex metabolic network of the FHP. Except for the recognition of its complexity, the metabolic flux network has not been well understood. Studies on biochemical reactions and metabolic flux network associated with the FHP in anaerobic fermentation system have only been drawn attention in recent years. This review summarizes the biochemical reactions taking place in the metabolic network of FHP. We discuss how the key operation factors influence metabolism in the FHP process. Recently developed and applied technologies for metabolic flux analysis have been described. Future studies on the metabolic network to enhance fermentative hydrogen production by strict anaerobes are recommended. It is expected that this review can provide useful information in terms of fundamental knowledge and update technology for scientists and research engineers in the field of biological hydrogen production.     

Microalgae and Cyanobacteria: How Exploiting These Microbial Resources Can Address the Underlying Challenges Related to Food Sources and Sustainable Agriculture: A Review

For thousands of years, crop production has almost entirely depended on conventional agriculture. However, the reality is changing. The ever-growing population, global climate change, soil degradation and biotic/abiotic stresses are a growing threat to food production and security. Thus, sustainable alternatives to increase crop production for a population projected to reach 9.8 billion by 2050 are a major priority. In addition to vertical and soilless farming, innovative products based on bioresources, including plant growth stimulants, have been a target for sustainable food production. Such solutions have led to the exploitation of microorganisms, including microalgae and cyanobacteria as potential bioresources for food and plant biostimulant products. Microalgae (eukaryotic) and cyanobacteria (prokaryotic) are photosynthetic microorganisms with the capacity to synthesize a vast array of bioactive metabolites from atmospheric CO₂ and inorganic nutrients. The present review outlines the nutritional value of microalgae and cyanobacteria as alternative food resources. The potential aspects of microalgae and cyanobacteria as stabilizers of the net change in soil organic carbon (C) levels for reduced farmland degradation are also highlighted. The applications of microalgae and cyanobacteria as remedies for improved soil structure and fertility, and as enhancers of crop productivity and abiotic stress tolerance in agricultural settings are outlined. This review also discusses the co-cultivation of crops with microalgae or cyanobacteria in hydroponic systems to favor optimum root CO₂/O₂ levels for optimized crop production.

     

DRUM: A New Framework for Metabolic Modeling under Non-Balanced Growth. Application to the Carbon Metabolism of Unicellular Microalgae

Metabolic modeling is a powerful tool to understand, predict and optimize bioprocesses, particularly when they imply intracellular molecules of interest. Unfortunately, the use of metabolic models for time varying metabolic fluxes is hampered by the lack of experimental data required to define and calibrate the kinetic reaction rates of the metabolic pathways. For this reason, metabolic models are often used under the balanced growth hypothesis. However, for some processes such as the photoautotrophic metabolism of microalgae, the balanced-growth assumption appears to be unreasonable because of the synchronization of their circadian cycle on the daily light. Yet, understanding microalgae metabolism is necessary to optimize the production yield of bioprocesses based on this microorganism, as for example production of third-generation biofuels. In this paper, we propose DRUM, a new dynamic metabolic modeling framework that handles the non-balanced growth condition and hence accumulation of intracellular metabolites. The first stage of the approach consists in splitting the metabolic network into sub-networks describing reactions which are spatially close, and which are assumed to satisfy balanced growth condition. The left metabolites interconnecting the sub-networks behave dynamically. Then, thanks to Elementary Flux Mode analysis, each sub-network is reduced to macroscopic reactions, for which simple kinetics are assumed. Finally, an Ordinary Differential Equation system is obtained to describe substrate consumption, biomass production, products excretion and accumulation of some internal metabolites. DRUM was applied to the accumulation of lipids and carbohydrates of the microalgae Tisochrysis lutea under day/night cycles. The resulting model describes accurately experimental data obtained in day/night conditions. It efficiently predicts the accumulation and consumption of lipids and carbohydrates.     

Application of Photoautotrophic Suspension Cultures in Plant Science

Whereas most plant suspension cultures are grown heterotrophically in the presence of sugars, a limited number of photoautotrophic cultures have been established which are able to grow with CO₂ as the sole carbon source. Photoautotrophic cultures are useful to address various aspects of photosynthesis, source-sink regulation, nitrogen metabolism, production of secondary metabolites, and defence responses. The homogenous populations of these cultures provide an ideal and sensitive system to obtain reproducible results. The availability of an increasing number of photoautotrophic cultures from different economically important species provides the basis also for practical applications.