Search

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d‐mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast‐growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two‐step pathway by cloning genes for mannitol‐1‐phosphate dehydrogenase (mtlD) and mannitol‐1‐phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, Prbc225, PcpcB300, PcpcBm1, PrbcLm17, and PrbcLm15. The strains were tested under the "switch conditions," where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing Prbc225‐mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD730) was exhibited by the engineered strain of PCC 7942 expressing PcpcB300‐mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria. [ABSTRACT FROM AUTHOR]

Mitigating greenhouse-gas emissions, of which fossil-derived carbon dioxide (CO 2) is the dominant component, is becoming increasingly imperative. One of the tools for lowering the demand for fossil carbon is cultivation of microalgae, which are fast-growing photosynthetic microorganisms that utilize sunlight for energy and CO 2 as their carbon source. In addition, microalgae can provide feedstock to replace fossil sources, particularly for transportation fuels. In open and closed microalgal cultivating systems (also called open ponds and photobioreactors, respectively), CO 2 can be sparged into the culture medium through a gas distributor; CO 2 molecules diffuse through the gas-liquid interface and dissolve into the culture medium, from which they can be taken up for the biosynthesis of microalgal cells. Due to the modest solubility of CO 2 in water, optimal design and operating variables (e.g., inlet gas flow rate, sparger characteristics, CO 2 concentration in the inlet gas, and the height of a PBR or sump) are required to increase the CO 2 mass transfer rate into the medium and, consequently, CO 2 uptake and biomass productivity. The concepts and phenomena discussed in this work apply to photobioreactors and open ponds that are sparged with CO 2. This review systematically evaluates how the key design and operating variables affect bubble behavior and the rate of CO 2 delivery into the medium. The review also addresses advanced strategies that are being employed to increase the rate of CO 2 transfer, but with lower costs than with sparging. [Display omitted] • CO 2 management is critical during microalgal cultivation in photobioreactors (PBRs). • Inlet CO 2 concentration and gas flow rate affect CO 2 mass transfer. • Sparger design and sparging style determine effective CO 2 delivery to the system. • Carbon utilization efficiency (CUE) is dictated by the pH management in PBRs. • CUE can be improved through membrane carbonation or by using carbonic anhydrases. [ABSTRACT FROM AUTHOR]

Abstract Heparosan is an unsulfated polysaccharide potentially important for its wide range of cosmetic and pharmaceutical applications, particularly as the precursor for the extensively used anticoagulant, heparin. Generally sourced from animals, commercially available heparin may encounter various immunological and contamination risks. Thus, safe and sustainable microbial platforms could serve as an alternative heparin source. Synechococcus , due to their fast photoautotrophic growth, strong sugar phosphate metabolisms and generally regarded as safe (GRAS) nature, may serve as photo-biorefineries for manufacturing heparosan. In this study, we have synthesized an integrative plasmid pUPm48 for cloning galU and PmHS2 genes in Synechococcus elongatus PCC 7942. The engineered recombinants (pgp7942) exhibited significant production of heparosan under different culture conditions, where the products were present in both supernatant and cell biomass. The maximum yield of 0.7 ± 0.2 μg/g-DCW (dry cell weight) and a titer of 2.8 ± 0.3 μg/L was achieved by pgp7942 under shake flask and continuous light conditions. Large scale plastic-bag cultures with natural diurnal light exhibited heparosan production of 0.5 μg/g-DCW with a titer of 0.44 μg/L. The analysis also found PCC 7942 encodes a promiscuous uridyltransferase for UDP-glucose synthesis and naturally produces multiple glycosaminoglycans including chondroitin sulfate (CS). This study demonstrates for the first-time cyanobacteria as a promising photoautotrophic refinery for producing a high-value polysaccharide commonly from animals. Highlights • PCC 7942 can naturally synthesize multiple glycosaminoglycans including chondroitin sulfate (CS). • Heparosan synthase from pathogenic P. multocida was expressed in S. elongatus PCC 7942. • Heparosan was retained intracellularly and also secreted into the extracellular medium. • Under photoautotrophic conditions, 2.8 μg/L of heparosan was produced. [ABSTRACT FROM AUTHOR]

Abstract Cyanobacteria are globally recognized as potential photosynthetic platform cell factories for production of value-added chemicals. Carotenoids are industrially important fine chemicals used in food, pharmaceutical and health-care products. Zeaxanthin has emerged as a high value xanthophyll carotenoid that exhibits superior antioxidant properties over many other carotenoids. Traditionally sourced from plants like marigold flowers wherein, zeaxanthin co-occurs with lutein. Therefore, there is a need for isolated production system for zeaxanthin on account of difficulties in separation of zeaxanthin from lutein due to their very similar molecular structures. Synechococcus elongatus PCC 7942 (PCC 7942) is a cyanobacterium that is known to synthesize zeaxanthin as one of the predominant cellular carotenoids and does not possess pathway genes for lutein production. In order to construct a system capable of industrial production of zeaxanthin, we genetically modified PCC 7942 (Synechococcus 79R48) to improve β‑carotene flux towards zeaxanthin synthesis by cloning CrtR (β‑carotene oxygenase) gene from Synechococcus elongatus PCC 7002 through homologous recombination. This strategy effectively enhanced the yield (mg/g DCW) of zeaxanthin in the transformants. Moreover, to increase zeaxanthin titer (mg/L), an operon construct was synthesized where a heterologous gene GalP (Hexose-H+ symporter) from E. coli MG1655 was cloned downstream of CrtR (Synechococcus 79RG48) which facilitated inherently obligate photoautotrophic WT PCC 7942 cells to uptake extracellular glucose in transformants, thereby increasing biomass productivity. Autotrophically grown cells yielded 9.02 ± 1.10 mg/g DCW of zeaxanthin while under mixotrophy the yield was 8.09 ± 0.19 mg/g DCW, which was 2-fold improvement over the wild type. The volumetric productivities of the transformants were 1.18 ± 0.17 mg/L·d and 1.8 ± 0.06 mg/L·d under autotrophic and mixotrophic conditions, respectively while WT produced 0.58 ± 0.02 mg/L·d. The work was successfully able to demonstrate that the modifications resulted in, enhanced production of lutein-free zeaxanthin in the cyanobacterial system. Highlights • Strategy for microbial production of lutein free-zeaxanthin through cyanobacterial genetic engineering • Cyanobacteria are suitable candidates for biological production of lutein-free zeaxanthin. • Single-gene flux improvement strategy for over-production of zeaxanthin • Improved quantum of light energy utilization for carotenoid biosynthesis through mixotrophy under natural light [ABSTRACT FROM AUTHOR]

Alkanes are high-energy hydrocarbons that are foreseen as next generation biofuels. Cyanobacteria are known to naturally synthesize C15-C19 alkanes; however, the titers are too low to make this a commercially viable process. Therefore, to leverage these photosynthetic platforms for improved alkane production, here we engineered three novel isolates of Synechococcus elongatus PCC 11801, PCC 11802, and IITB6. The two gene AAR-ADO alkane biosynthesis pathway was constructed by cloning the genes for acyl-ACP reductase (aar) and aldehyde deformylating oxygenase (ado) from S. elongatus PCC 7942 under the regulation of P rbcL promoter from PCC 7942 and native promoters from PCC 11801 such as P cpcB300 , P psbAI , and P psbAIII . The genes were separately cloned under two different promoters, creating a library of the engineered strains. The results indicated that the engineered strains of novel S. elongatus isolates produced significantly higher amounts of alkanes than the model strain PCC 7942. The highest alkane yield achieved was 4.1 mg/gDCW in BG-11, while the highest titer was 31.5 mg/L in 5X BG-11, with an engineered IITB6 strain (P cpcB300 :aar:T rrnB ::P rbcL :ado:T Lac ). Overall, the study highlights the potential of newly isolated S. elongatus strains as efficient alkane production platforms., (© 2024 American Institute of Chemical Engineers.)

Engineered cyanobacteria are attractive hosts for the phototrophic conversion of CO 2 to chemicals. Synechococcus elongatus PCC11801, a novel, fast-growing, and stress-tolerant cyanobacterium, has the potential to be a platform cell factory, and hence, it necessitates the development of a synthetic biology toolbox. Considering the widely followed cyanobacterial engineering strategy of chromosomal integration of heterologous DNA, it is of interest to discover and validate new chromosomal neutral sites (NSs) in this strain. To that end, global transcriptome analysis was performed using RNA Seq under the conditions of high temperature (HT), carbon (HC), and salt (HS) and ambient growth conditions. We found upregulation of 445, 138, and 87 genes and downregulation of 333, 125, and 132 genes, under HC, HT, and HS, respectively. Following nonhierarchical clustering, gene enrichment, and bioinformatics analysis, 27 putative NSs were predicted. Six of them were experimentally tested, and five showed confirmed neutrality, based on unaltered cell growth. Thus, global transcriptomic analysis was effectively exploited for NS annotation and would be advantageous for multiplexed genome editing.