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      Engineered cyanobacteria with additional overexpression of selected Calvin-Benson-Bassham enzymes show further increased ethanol production

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          Abstract

          Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803.

          The ethanol production of the strains overexpressing two CBB enzymes (FBA ​+ ​TK, FBP/SBPase ​+ ​FBA or FBP/SBPase ​+ ​TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

          Highlights

          • . Effects of CBB enzymes expression on ethanol yield in Synechocystis PCC 6803 analyzed.

          • . Increased ethanol production when overexpression two selected CBB enzymes.

          • . Highest ethanol production in strain overexpressing a combination of FBA and TK.

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          Most cited references35

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          Purification and properties of unicellular blue-green algae (order Chroococcales).

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            Optimizing the distribution of resources between enzymes of carbon metabolism can dramatically increase photosynthetic rate: a numerical simulation using an evolutionary algorithm.

            The distribution of resources between enzymes of photosynthetic carbon metabolism might be assumed to have been optimized by natural selection. However, natural selection for survival and fecundity does not necessarily select for maximal photosynthetic productivity. Further, the concentration of a key substrate, atmospheric CO(2), has changed more over the past 100 years than the past 25 million years, with the likelihood that natural selection has had inadequate time to reoptimize resource partitioning for this change. Could photosynthetic rate be increased by altered partitioning of resources among the enzymes of carbon metabolism? This question is addressed using an "evolutionary" algorithm to progressively search for multiple alterations in partitioning that increase photosynthetic rate. To do this, we extended existing metabolic models of C(3) photosynthesis by including the photorespiratory pathway (PCOP) and metabolism to starch and sucrose to develop a complete dynamic model of photosynthetic carbon metabolism. The model consists of linked differential equations, each representing the change of concentration of one metabolite. Initial concentrations of metabolites and maximal activities of enzymes were extracted from the literature. The dynamics of CO(2) fixation and metabolite concentrations were realistically simulated by numerical integration, such that the model could mimic well-established physiological phenomena. For example, a realistic steady-state rate of CO(2) uptake was attained and then reattained after perturbing O(2) concentration. Using an evolutionary algorithm, partitioning of a fixed total amount of protein-nitrogen between enzymes was allowed to vary. The individual with the higher light-saturated photosynthetic rate was selected and used to seed the next generation. After 1,500 generations, photosynthesis was increased substantially. This suggests that the "typical" partitioning in C(3) leaves might be suboptimal for maximizing the light-saturated rate of photosynthesis. An overinvestment in PCOP enzymes and underinvestment in Rubisco, sedoheptulose-1,7-bisphosphatase, and fructose-1,6-bisphosphate aldolase were indicated. Increase in sink capacity, such as increase in ADP-glucose pyrophosphorylase, was also indicated to lead to increased CO(2) uptake rate. These results suggest that manipulation of partitioning could greatly increase carbon gain without any increase in the total protein-nitrogen investment in the apparatus for photosynthetic carbon metabolism.
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              Life and the evolution of Earth's atmosphere.

              Harvesting light to produce energy and oxygen (photosynthesis) is the signature of all land plants. This ability was co-opted from a precocious and ancient form of life known as cyanobacteria. Today these bacteria, as well as microscopic algae, supply oxygen to the atmosphere and churn out fixed nitrogen in Earth's vast oceans. Microorganisms may also have played a major role in atmosphere evolution before the rise of oxygen. Under the more dim light of a young sun cooler than today's, certain groups of anaerobic bacteria may have been pumping out large amounts of methane, thereby keeping the early climate warm and inviting. The evolution of Earth's atmosphere is linked tightly to the evolution of its biota.
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                Author and article information

                Contributors
                Journal
                Metab Eng Commun
                Metab Eng Commun
                Metabolic Engineering Communications
                Elsevier
                2214-0301
                11 January 2021
                June 2021
                11 January 2021
                : 12
                : e00161
                Affiliations
                [1]Microbial Chemistry, Department of Chemistry-Ångström, Uppsala University, Box 523, SE-751 20, Uppsala, Sweden
                Author notes
                []Corresponding author. Peter.Lindblad@ 123456kemi.uu.se
                Article
                S2214-0301(21)00001-8 e00161
                10.1016/j.mec.2021.e00161
                7820548
                33520653
                5b6d3b47-53cd-4bf8-bd37-586711e02b7c
                © 2021 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 22 September 2020
                : 21 December 2020
                : 6 January 2021
                Categories
                Special issue on Engineering Cyanobacteria edited by Peter Lindblad and Jens Krömer

                cyanobacteria,calvin-benson-bassham,ethanol,fbp/sbpase,tk,fba,carbon fixation

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