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Cyanobacteria, a group of photoautotrophic prokaryotes, are attractive hosts for the sustainable production of chemicals from carbon dioxide and sunlight. However, the rates, yields, and titers have remained well below those needed for commercial deployment. We argue that the following areas will be central to the development of cyanobacterial cell factories: engineered and well-characterized host strains, model-guided pathway design, and advanced synthetic biology tools. Although several foundational studies report improved strain properties, translational research will be needed to develop engineered hosts and deploy them for metabolic engineering. Further, the recent developments in metabolic modeling and synthetic biology of cyanobacteria will enable nimble strategies for strain improvement with the complete cycle of design, build, test, and learn.

Cyanobacteria are attractive model organisms for studies of photosynthesis and diurnal metabolism and as hosts for photoautotrophic production of chemicals. Exposure to bright light, environmental pollutants, and diurnal lifestyle of these prokaryotes may result in significant oxidative stress. Glutathione is a widely studied gamma-glutamyl peptide that plays a key role in managing oxidative stress and detoxification of xenobiotics in cyanobacteria. The functional role and biosynthetic pathways of this tripeptide have been studied in detail in various phyla, including cyanobacteria. However, other γ-glutamyl peptides remain largely unexplored. We use an integrated approach to identify a number of γ-glutamyl peptides based on signature mass fragments and mass shifts in them in 13 C and 15 N enriched metabolite extracts. The newly identified compounds include γ-glutamyl dipeptides and derivatives of glutathione. Carbon backbones of the former turn over much faster than that of glutathione suggesting that they follow a distinct biosynthetic pathway. Further, transients of isotopic 13 C enrichment show positional labeling in these peptides which allow us to delineate between the alternative biosynthetic pathways. Importantly, the amino acid of γ-glutamyl dipeptides shows much faster turnover compared to the glutamate moiety. The significant accumulation of γ-glutamyl dipeptides under slow-growth conditions combined with the results from dynamic 13 C labeling suggests that these compounds may act as reservoirs of amino acids in cyanobacteria.

Cyanobacteria, a group of photosynthetic prokaryotes, are attractive hosts for biotechnological applications. It is envisaged that future biorefineries will deploy engineered cyanobacteria for the conversion of carbon dioxide to useful chemicals via light-driven, endergonic reactions. Fast-growing, genetically amenable, and stress-tolerant cyanobacteria are desirable as chassis for such applications. The recently reported strains such as Synechococcus elongatus UTEX 2973 and PCC 11801 hold promise, but additional strains may be needed for the ongoing efforts of metabolic engineering. Here, we report a novel, fast-growing, and naturally transformable cyanobacterium, S. elongatus PCC 11802, that shares 97% genome identity with its closest neighbor S. elongatus PCC 11801. The new isolate has a doubling time of 2.8 h at 1% CO2, 1000 µmole photons.m−2.s−1 and grows faster under high CO2 and temperature compared to PCC 11801 thus making it an attractive host for outdoor cultivations and eventual applications in the biorefinery. Furthermore, S. elongatus PCC 11802 shows higher levels of key intermediate metabolites suggesting that this strain might be better suited for achieving high metabolic flux in engineered pathways. Importantly, metabolite profiles suggest that the key enzymes of the Calvin cycle are not repressed under elevated CO2 in the new isolate, unlike its closest neighbor.

Cyanobacteria are attractive model organisms for the study of photosynthesis and diurnal metabolism and as hosts for photoautotrophic production of chemicals. Exposure to bright light or environmental pollutants and a diurnal lifestyle of these prokaryotes may result in significant oxidative stress. Glutathione is a widely studied γ-glutamyl peptide that plays a key role in managing oxidative stress and detoxification of xenobiotics in cyanobacteria. The functional role and biosynthesis pathways of this tripeptide have been studied in detail in various phyla, including cyanobacteria. However, other γ-glutamyl peptides remain largely unexplored. We use an integrated approach to identify a number of γ-glutamyl peptides based on signature mass fragments and mass shifts in them in

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 upregulated, and 1715 downregulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins, such as vitellogenins, extensin, laminin, tropomyosin, troponin C, and flightin. Majority of the differentially expressed genes were categorized under molecular functions followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide, and energy metabolic pathways, the endocytotic pathway, and the defense-related pathways. Differential regulation of genes associated with the key pathways might favour CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02641-x.

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 &beta, 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 &mu, mole/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.

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits the citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Application of insecticides to manage the psyllid vectors and disease is the most common practice. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 up-regulated, and 1715 down-regulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins. Majority of the differentially expressed genes were categorized under molecular function followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide and energy metabolic pathways, the endocytotic pathway and the defense-related pathways. Differential regulation of genes associated with the key pathways might favors CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas.

Background Cyanobacteria, 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. Results Building 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. Conclusion While 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.

Recently, the sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) method coupled with liquid chromatography has been demonstrated for the quantification of isotopic

Accurate quantification of mass isotopolog distribution (MID) of intracellular metabolites is a key requirement for

Accurate quantification of mass isotopolog distribution (MID) of intracellular metabolites is a key requirement for 13C metabolic flux analysis (13C-MFA). Liquid chromatography coupled with mass spectrometry (LC/MS) has emerged as a frontrunner technique that combines two orthogonal separation strategies. While metabolomics requires separation of monoisotopic peaks, 13C-MFA imposes additional demands for chromatographic separation as isotopologs of metabolites significantly add to the number of analytes. In this protocol chapter, we discuss two liquid chromatography methods, namely, reverse phase ion-pairing and hydrophilic interaction chromatography (HILIC) that together can separate a wide variety of metabolites that are typically used for 13C metabolic flux analysis.

Recently, the sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) method coupled with liquid chromatography has been demonstrated for the quantification of isotopic 13C enrichment in a large number of cellular metabolites and fragments. SWATH, a data-independent acquisition (DIA) method, alleviates the need for data deconvolution and shows greater accuracy in the quantification of low abundance isotopologs of fragments thereby resulting in a lower systematic error. Here we provide a detailed protocol for the design of Q1 mass isolation windows and the post-acquisition data analysis with emphasis on the untargeted nature of SWATH.

Additional file 1. Additional figures and tables.

In the recent years, solar powered cyanobacteria have emerged as promising cell factories for the production of biofuels, bioplastics and platforms chemicals. This is due to their inherent ability to fix carbon dioxide, fast growth rate, minimal nutrient requirements and relative ease of genetic manipulation. However, significant improvement in productivity will be required before the cyanobacterial cell factories can be commercially deployed. Eventual success will depend on the availability of synthetic biology tools such as well characterized promoters, ribosomal binding sites, terminators and genome editing methods. In the present chapter, we discuss the different synthetic biology-based approaches that have been used to engineer model strains of cyanobacteria. The strain development strategies are expected to be dependent on the desired product and the corresponding precursor from the central carbon pathway. Finally, an outlook on the future developments that could expedite the commercialization of cyanobacteria for renewable biotechnology has been suggested.

Accurate quantification of mass isotopologue distribution (MID) of metabolites is a prerequisite for 13C-metabolic flux analysis. Currently used mass spectrometric (MS) techniques based on multiple reaction monitoring (MRM) place limitations on the number of MIDs that can be analyzed in a single run. Moreover, the deconvolution step results in amplification of error. Here, we demonstrate that SWATH MS/MS, a data independent acquisition (DIA) technique allows quantification of a large number of precursor and product MIDs in a single run. SWATH sequentially fragments all precursor ions in stacked mass isolation windows. Co-fragmentation of all precursor isotopologues in a single SWATH window yields higher sensitivity enabling quantification of MIDs of fragments with low abundance and lower systematic and random errors. We quantify the MIDs of 53 precursor and product ions corresponding to 19 intracellular metabolites from a dynamic 13C-labeling of a model cyanobacterium, Synechococcus sp. PCC 7002. The use ...

Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST-13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc.

13C Metabolic Flux Analysis (13C-MFA) is a powerful tool for quantification of carbon flux distribution in metabolic pathways. However, the requirement to obtain accurate labeling patterns, especially for compounds with low abundance, poses a challenge. Chromatographic separation and high sensitivity of the modern mass spectrometers (MS) alleviate this problem to a certain extent. However, the presence of derivatives such as in-source fragments, multimer ion adducts, and multiply charged ions result in reduced intensity of the molecular ion. While multimer ion adducts have been reported in the field of metabolomics, their presence is considered undesirable in quantitative studies. Here, we demonstrate a novel application of dimer ion adducts in calculating the mass isotopologue distribution (MIDs) of the corresponding monomer ions for public domain and in-house generated datasets comprising of 13C-labeling time-course experiments. Out of the 100 standard compounds analyzed, we could detect multimer ion adducts in 24 of the intermediate metabolites. Further, a subset of these multimer ions were detected in all the biological samples analyzed. Majority of these ion adducts were either not detected in the original study or labeled as a putative features. Regression analysis was performed to estimate the monomer MIDs from those of the dimer. This resulted in accurate estimation regardless of the biological system, chromatographic method, the MS hardware, or the relative abundance of the dimer ion. We argue that this analysis may be useful in cases where satisfactory data cannot be extracted from the chromatographic peaks of the monomer ions.

The environmental considerations attributing to the escalation of carbon dioxide emissions have raised alarmingly. Consequently, the concept of sequestration and biological conversion of CO2 by photosynthetic microorganisms is gaining enormous recognition. In this study, in an attempt to discern the synergistic CO2 tolerance mechanisms, metabolic responses to increasing CO2 concentrations were determined for Synechococcus elongatus PCC 11801, a fast-growing, novel freshwater strain, using quantitative proteomics. The protein expression data revealed that the organism responded to elevated CO2 by not only regulating the cellular transporters involved in carbon-nitrogen uptake and assimilation but also by inducing photosynthesis, carbon fixation and glycolysis. Several components of photosynthetic machinery like photosystem reaction centers, phycobilisomes, cytochromes, etc. showed a marked up-regulation with a concomitant downshift in proteins involved in photoprotection and redox maintenance. Additionally, enzymes belonging to the TCA cycle and oxidative pentose phosphate pathway exhibited a decline in their expression, further highlighting that the demand for reduced cofactors was fulfilled primarily through photosynthesis. The present study brings the first-ever comprehensive assessment of intricate molecular changes in this novel strain while shifting from carbon-limited to carbon-sufficient conditions and may pave the path for future host and pathway engineering for production of sustainable fuels through efficient CO2 capture.

13C Metabolic Flux Analysis (13C-MFA) involves the quantification of isotopic enrichment in cellular metabolites and fitting the resultant data to the metabolic network model of the organism. Coverage and resolution of the resultant flux map depends on the total number of metabolites and fragments in which 13C enrichment can be quantified accurately. Experimental techniques for tracking 13C enrichment are evolving rapidly and large volumes of data are now routinely generated through the use of Liquid Chromatography coupled with High-Resolution Mass Spectrometry (HR-LC/MS). Therefore, the current manuscript is focused on the challenges in high-throughput analyses of such large datasets. Current 13C-MFA studies often have to rely on the targeted quantification of a small subset of metabolites, thereby leaving a large fraction of the data unexplored. A number of public domain software tools have been reported in recent years for the untargeted quantitation of isotopic enrichment. However, the suitability of their application across diverse datasets has not been investigated. Here, we test the software tools X13CMS, DynaMet, geoRge, and HiResTEC with three diverse datasets. The tools provided a global, untargeted view of 13C enrichment in metabolites in all three datasets and a much-needed automation in data analysis. Some inconsistencies were observed in results obtained from the different tools, which could be partially ascribed to the lack of baseline separation and potential mass conflicts. After removing the false positives manually, isotopic enrichment could be quantified reliably in a large repertoire of metabolites. Of the software tools explored, geoRge and HiResTEC consistently performed well for the untargeted analysis of all datasets tested., Graphical abstract Image 1, Highlights • X13CMS, DynaMet, geoRge and HiResTEC were evaluated for untargeted and targeted analysis. • These software tools provided a quick global view of 13C enrichment in the metabolome. • Extensive parameter optimization and manual curation of results were necessary. • geoRge and HiResTEC worked well for untargeted analyses of dynamic labeling datasets.

A key requirement for C-13 Metabolic flux analysis (C-13-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The C-13 isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in C-13-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801. While minor changes in extraction solvent resulted in dramatic changes in the extraction efficiency of a number of compounds, certain metabolites such as amino acids and organic acids were adequately extracted in all the solvent systems tested. Overall, we present a new improved method for extraction using a methanol-chloroform-NH4OH system. Our method improves the extraction of polar compounds such as sugar phosphates, bisphosphates, that are central to C-13-MFA studies.

Cyanobacteria provide an interesting platform for biotechnological applications due to their efficient photoautotrophic growth, amenability to genetic engineering and the ability to grow on non-arable land. An ideal industrial strain of cyanobacteria would need to be fast growing and tolerant to high levels of temperature, light, carbon dioxide, salt and be naturally transformable. In this study, we report Synechococcus elongatus PCC 11801, a strain isolated from India that fulfills these requirements. The physiological and biochemical characteristics of PCC 11801 under carbon and light-limiting conditions were investigated. PCC 11801 shows a doubling time of 2.3 h, that is the fastest growth for any cyanobacteria reported so far under ambient CO2 conditions. Genome sequence of PCC 11801 shows genome identity of ~83% with its closest neighbors Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973. The unique attributes of PCC 11801 genome are discussed in light of the physiological characteristics that are needed in an industrial strain. The genome of PCC 11801 shows several genes that do not have homologs in neighbor strains PCC 7942 and UTEX 2973, some of which may be responsible for adaptation to various abiotic stresses. The remarkably fast growth rate of PCC 11801 coupled with its robustness and ease of genetic transformation makes it an ideal candidate for the photosynthetic production of fuels and chemicals.

Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary