Increased triacylglycerol production in Rhodococcus opacus by overexpressing transcriptional regulators.

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the ß-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Advances in ligand-specific biosensing for structurally similar molecules.

The specificity of biological systems makes it possible to develop biosensors targeting specific metabolites, toxins, and pollutants in complex medical or environmental samples without interference from structurally similar compounds. For the last two decades, great efforts have been devoted to creating proteins or nucleic acids with novel properties through synthetic biology strategies. Beyond augmenting biocatalytic activity, expanding target substrate scopes, and enhancing enzymes' enantioselectivity and stability, an increasing research area is the enhancement of molecular specificity for genetically encoded biosensors. Here, we summarize recent advances in the development of highly specific biosensor systems and their essential applications. First, we describe the rational design principles required to create libraries containing potential mutants with less promiscuity or better specificity. Next, we review the emerging high-throughput screening techniques to engineer biosensing specificity for the desired target. Finally, we examine the computer-aided evaluation and prediction methods to facilitate the construction of ligand-specific biosensors.

Upcycling of poly(ethylene terephthalate) to produce high-value bio-products.

More than 70 million tons of poly(ethylene terephthalate) (PET) are manufactured worldwide every year. The accumulation of PET waste has become a global pollution concern, motivating the urgent development of technologies to valorize post-consumer PET. The development of chemocatalytic and enzymatic approaches for depolymerizing PET to its corresponding monomers opens up new opportunities for PET upcycling through biological transformation. Here, we identify Rhodococcus jostii strain PET (RPET) that can directly use PET hydrolysate as a sole carbon source. We also investigate the potential of RPET to upcycle PET into value-added chemicals, using lycopene as a proof-of-concept product. Through rational metabolic engineering, we improve lycopene production by more than 500-fold over that of the wild type. In addition, we demonstrate the production of approximately 1,300 µg/L lycopene from PET by cascading this strain with PET alkaline hydrolysis. This work highlights the great potential of biological conversion as a means of achieving PET upcycling.

Deciphering the transcriptional regulation of the catabolism of lignin-derived aromatics in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 has considerable potential as a platform for valorizing lignin due to its innate "biological funneling" pathways. However, the transcriptional regulation of the aromatic catabolic pathways and the mechanisms controlling aromatic catabolic operons in response to different aromatic mixtures are still underexplored. Here, we identified and studied the transcription factors for aromatic degradation using GFP-based sensors and comprehensive deletion analyses. Our results demonstrate that the funneling pathways for phenol, guaiacol, 4-hydroxybenzoate, and vanillate are controlled by transcriptional activators. The two different branches of the ß-ketoadipate pathway, however, are controlled by transcriptional repressors. Additionally, promoter activity assays revealed that the substrate hierarchy in R. opacus may be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways. These results provide clues to clarify the molecule-level mechanisms underlying the complex regulation of aromatic catabolism, which facilitates the development of R. opacus as a promising chassis for valorizing lignin.

Rewiring the Metabolic Network to Increase Docosahexaenoic Acid Productivity in Crypthecodinium cohnii by Fermentation Supernatant-Based Adaptive Laboratory Evolution.

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate Crypthecodinium cohnii is a good candidate to produce high-quality DHA. To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of DHA-producing C. cohnii, fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87 and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87 and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE. In addition, a comparative proteomic analysis identified 11,106 proteins and 910 differentially expressed proteins, including six stress-responsive proteins, as well as the up- and downregulated pathways in FS280 that might contribute to its improved cell growth and DHA accumulation. Our study demonstrated that FS-ALE could be a valuable solution to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation of heterotrophic microalgae.

Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions.

In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.

An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus.

Rhodococcus opacus is a nonmodel bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in nonmodel organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool's utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. Our CRISPRi tool will be useful to facilitate functional studies of this nonmodel organism and engineer this promising microbial chassis for lignin valorization.

Tailoring cyanobacteria as a new platform for highly efficient synthesis of astaxanthin.

With the ability to recycle CO2 into value-added chemicals, cyanobacteria have been considered as renewable microbial cell factories. Astaxanthin, a highly valued carotenoid with potent antioxidant activity, could be beneficial to human health. Astaxanthin biosynthesis in engineered chassis has been achieved previously, but it generated a relatively low yield. Here, we successfully constructed a highly efficient astaxanthin biosynthetic pathway in cyanobacterium Synechocystis sp. PCC 6803, and achieved more than a 500-fold increase in astaxanthin production via stepwise reconstruction of the biosynthetic pathway and rational rewiring of the endogenous metabolism. The engineered strain produced up to 29.6 mg/g of astaxanthin (dry cell weight), which is the highest yield reported in the engineered chassis to date. Moreover, multi-omics analyses revealed that establishing a high astaxanthin flux may enhance photosynthesis and central metabolism in the engineered strain to compensate for the depleted pigments, which could be valuable for astaxanthin overproduction. This study presents a novel alternative for high-efficiency biosynthesis of astaxanthin directly from CO2.

Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO2.

BACKGROUND: Cyanobacterial carbohydrates, such as sucrose, have been considered as potential renewable feedstock to support the production of fuels and chemicals. However, the separation and purification processes of these carbohydrates will increase the production cost of chemicals. Co-culture fermentation has been proposed as an efficient and economical way to utilize these cyanobacterial carbohydrates. However, studies on the application of co-culture systems to achieve green biosynthesis of platform chemicals are still rare. RESULTS: In this study, we successfully achieved one-step conversion of sucrose derived from cyanobacteria to fine chemicals by constructing a microbial consortium consisting of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 and Escherichia coli to sequentially produce sucrose and then the platform chemical 3-hydroxypropionic acid (3-HP) from CO2 under photoautotrophic growth conditions. First, efforts were made to overexpress the sucrose permease-coding gene cscB under the strong promoter P cpc560 in S. elongatus UTEX 2973 for efficient sucrose secretion. Second, the sucrose catabolic pathway and malonyl-CoA-dependent 3-HP biosynthetic pathway were introduced into E. coli BL21 (DE3) for heterologous biosynthesis of 3-HP from sucrose. By optimizing the cultivation temperature from 37 to 30 °C, a stable artificial consortium system was constructed with the capability of producing 3-HP at up to 68.29 mg/L directly from CO2. In addition, cell growth of S. elongatus UTEX 2973 in the consortium was enhanced, probably due to the quick quenching of reactive oxygen species (ROS) in the system by E. coli, which in turn improved the photosynthesis of cyanobacteria. CONCLUSION: The study demonstrated the feasibility of the one-step conversion of sucrose to fine chemicals using an artificial consortium system. The study also confirmed that heterotrophic bacteria could promote the cell growth of cyanobacteria by relieving oxidative stress in this microbial consortium, which further suggests the potential value of this system for future industrial applications.

Repeated fed-batch strategy and metabolomic analysis to achieve high docosahexaenoic acid productivity in Crypthecodinium cohnii.

BACKGROUND: Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC- and GC-MS metabolomic analyses. RESULTS: It was discovered that glucose concentration at 15-27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC- and GC-MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, L-tryptophan, L-threonine, L-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. CONCLUSIONS: The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Cellular engineering strategies toward sustainable omega-3 long chain polyunsaturated fatty acids production: State of the art and perspectives.

Long-chain polyunsaturated fatty acids (LC-PUFAs) especially ω-3 fatty acids provide significant health benefits for human beings. However, ω-3 LC-PUFAs cannot be synthesized de novo in mammals. Traditionally, ω-3 LC-PUFAs are extracted from marine fish, and their production depends on sea fishing, which has not met ever-increasing global demand. To address the challenges, innovative cellular engineering strategies need to be developed. In nature, many fungi and microalgae are rich in ω-3 LC-PUFAs, representing promising sources of ω-3 LC-PUFAs. The latest progress in developing new cellular engineering strategies toward sustainable ω-3 LC-PUFAs production using fungi and microalga has demonstrated that they can to some extent address the supply shortage. In this review, we critically summarize the recent progress in enhancing the productivity in various ω-3 LC-PUFAs-producing organisms, as well as the latest efforts of biosynthesizing PUFAs in heterogenous biosystems. In addition, we also provide future perspectives in developing genetic toolkits for LC-PUFAs producing microbes so that cut-edging biotechnology such as gene stacking and genome editing can be further applied to increase the productivity of ω-3 LC-PUFAs.

Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31.

BACKGROUND: Docosahexaenoic acid (DHA, C22:6) and odd-chain fatty acids (OCFAs, C15:0 and C17:0) have attracted great interest, since they have been widely used in food and therapeutic industries, as well as chemical industry, such as biodiesel production and improvement. The oil-producing heterotrophic microalgae Schizochytrium sp. 31 is one of main DHA-producing strains. Recently, it was found that Schizochytrium can also synthesize OCFAs; however, contents and titers of DHA and OCFAs in Schizochytrium are still low, which limit its practical application. RESULTS: In this study, we found that acetyl-CoA carboxylase suffered from a feedback inhibition by C16-CoA in Schizochytrium, and relief of the inhibition resulted in improved both lipid content and the ratio of OCFAs in total fatty acids. Based on this finding, a novel strategy for elevating both DHA and OCFAs contents was established. First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from Mortierella alpina, which encodes an elongase enzyme responsible for converting C16 into C18 fatty acids. After the above two-step engineering, contents of DHA and OCFAs were increased by 1.39- and 3.30-fold, reaching a level of 26.70 and 25.08% of dry cell weight, respectively, which are the highest contents reported so far for Schizochytrium. The titers of DHA and OCFAs were elevated by 1.08- and 2.57-fold, reaching a level of 3.54 and 3.32 g/L, respectively. Notably, the OCFAs titer achieved was 2.66-fold higher than the highest reported in Escherichia coli (1.25 g/L), implying potential value for industry application. To reveal the potential metabolic mechanism for the enhanced biosynthesis of both DHA and OCFAs, LC-MS metabolomic analysis was employed and the results showed that the pentose phosphate pathway and the glycolysis pathway were strengthened and intracellular propionyl-CoA concentration were also significantly increased in the engineered Schizochytrium, suggesting an increased supply of NADPH, acetyl-CoA, and propionyl-CoA for DHA and OCFAs accumulation. CONCLUSIONS: The discovery provides a new source of OCFAs production, and proposes a new strategy to improve contents and titers of both DHA and OCFAs in Schizochytrium. These will be valuable for improving commercial potential of Schizochytrium and guiding the engineering strategy in other fatty acids producing heterotrophic microalga.

Rewiring metabolic network by chemical modulator based laboratory evolution doubles lipid production in Crypthecodinium cohnii.

Dietary omega-3 long-chain polyunsaturated fatty acids docosahexaenoic acid (DHA, C22:6) can be synthesized in microalgae Crypthecodinium cohnii; however, its productivity is still low. Here, we established a new protocol termed as "chemical modulator based adaptive laboratory evolution" (CM-ALE) to enhance lipid and DHA productivity in C. cohnii. First, ACCase inhibitor sethoxydim based CM-ALE was applied to redirect carbon equivalents from starch to lipid. Second, CM-ALE using growth modulator sesamol as selection pressure was conducted to relive negative effects of sesamol on lipid biosynthesis in C. cohnii, which allows enhancement of biomass productivity by 30% without decreasing lipid content when sesamol was added. After two-step CM-ALE, the lipid and DHA productivity in C. cohnii was respectively doubled to a level of 0.046 g/L/h and 0.025 g/L/h in culture with addition of 1 mM sesamol, demonstrating that this two-step CM-ALE could be a valuable approach to maximize the properties of microalgae.

Direct Photosynthetic Production of Plastic Building Block Chemicals from CO2.

Hydroxy acids have attracted attention as building block chemicals due to their roles as precursors for the production of various pharmaceuticals, vitamins, antibiotics, and flavor compounds as well as monomers for biodegradable plastic polyesters. The current approach to hydroxy acid production relies on nonrenewable fossil resources such as petroleum for raw materials, raising issues such as the rising costs of starting materials and environmental incompatibility. Recently, synthetic biology approaches based on the rational design and reconstruction of new biological systems were implemented to produce chemicals from a variety of renewable substrates. In addition to research using heterotrophic organic carbon-dependent Escherichia coli or yeasts, photosynthetic microorganisms such as cyanobacteria possessing the ability to absorb solar radiation and fix carbon dioxide (CO2) as a sole carbon source have been engineered into a new type of microbial cell factory to directly produce hydroxy acids from CO2. In this chapter, recent progress regarding the direct photosynthetic production of three important hydroxy acids-3-hydroxypropionate (3-HP), 3-hydroxybutyrate (3-HB), and 3-hydroxyvalerate (3-HV)-from CO2 in cyanobacteria is summarized and discussed.

13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.

Toolboxes for cyanobacteria: Recent advances and future direction.

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral "microbial cell factories". Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO2, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.

Genetic Engineering of Crypthecodinium cohnii to Increase Growth and Lipid Accumulation.

In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO2 fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.

Comparative metabolomic analysis of Crypthecodinium cohnii in response to different dissolved oxygen levels during docosahexaenoic acid fermentation.

Oxygen supply is an important factor during Crypthecodinium cohnii fermentation for docosahexaenoic acid (DHA) production. However, few studies about the intrinsic correlation between dissolved oxygen (DO) and cellular metabolism have been reported. In this study, the responses of C. cohnii to different DO levels were evaluated. The results showed the growth and glucose consumption rates of C. cohnii were much higher under high oxygen supply condition. Furthermore, GC-MS based comparative metabolomic analysis was employed to discriminate the responsive metabolites associated with varying DO levels. The results showed the intermediates involved in glycolytic pathway and TCA cycle were up-regulated under high DO levels at exponential phase. At stationary phase, under high DO levels, metabolites involved in triacylglycerol metabolism were up-regulated, while the OPP pathway intermediate product ribose 5-phosphate was down-regulated. Together, these results provide useful insights into the functional metabolic relationship between DO levels and DHA production in C. cohnii.

Re-direction of carbon flux to key precursor malonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp. PCC 6803.

BACKGROUND: Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts. RESULTS: Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch. CONCLUSIONS: In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.

Screening and transcriptomic analysis of Crypthecodinium cohnii mutants with high growth and lipid content using the acetyl-CoA carboxylase inhibitor sethoxydim.

The heterotrophic microalga Crypthecodinium cohnii is well known for its lipid accumulation, with a high proportion of docosahexaenoic acid (DHA). In this study, we report a novel screening approach to obtain mutants of C. cohnii with high growth and lipid content using the acetyl-CoA carboxylase (ACCase) inhibitor sethoxydim. C. cohnii mutants were generated using atmospheric and room-temperature plasma (ARTP) and then screened for two rounds in media supplemented with sethoxydim. These efforts led to the identification of mutant M-1-2, which had 24.32% higher growth and 7.05% higher lipid content than the wild type, demonstrating the effectiveness of the sethoxydim-based screening. Consistently, the M-1-2 mutant displayed a 16.15% increase in ACCase enzymatic activity and 1.53-fold upregulation of its ACCase-encoding gene based on comparative ACCase activity analysis and transcriptomic analysis, respectively. In addition, transcriptomic analysis showed that transcripts involved in fatty acid biosynthesis, energy, central carbohydrate, and amino acid metabolism were upregulated in the mutant compared to the wild type.