Photosynthetic Co-production of Succinate and Ethylene in a Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801

Abstract

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 β 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 μmole/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.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle

“…Here we present an LC-MS/MS-based, large-scale metabolite profiling of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 (henceforth S. elongatus PCC 11801) in a diurnal sine LD cycle. S. elongatus PCC 11801 is a promising candidate for biotechnological applications owing to its faster growth, tolerance to high light and temperature, and genetic amenability (Jaiswal et al, 2018b(Jaiswal et al, , 2020bSengupta et al, 2020aSengupta et al, , 2020b. We find dramatic intra-day changes in the inventory of sugar bisphosphates, sugar phosphates, nucleotides, sugar nucleotides, organic acids, amino acids, and hitherto unexplored g-glutamyl dipeptides.…”

Comparative analysis of cyanobacteria species reveals a novel guanidine-degrading enzyme that controls genomic stability of ethylene-producing strains

“…1a,b, 5a, 6), our discovery of the guanidine-degrading activity of Sll1077 was leveraged to generate a derivative strain of Synechococcus 7942 that exhibits enhanced genomic stability and stable high-level production of ethylene in prolonged culture, which has not been achieved in prior studies (Fig. 6, S5) [23][24][25]41 . It is noteworthy that co-expression of Sll1077 with EFE substantially attenuate, but does not completely eliminate, the accumulation of guanidine in cultures of the engineered Synechococcus GD-EFE7942 strain (Fig.…”

“…PCC 6803 (hereafter Synechocystis 6803), have been able to accommodate stable, high-level expression of EFE and thereby sustain enhanced production of ethylene [17][18][19][20][21][22] . Other species, such as cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942) and Synechococcus elongatus PCC 11801 (hereafter Synechococcus 11801), however, have not been able to tolerate high-level expression of EFE, and the recombinant strains suffered severe growth inhibition [23][24][25] that was rescued by spontaneous chromosomal mutations that abolished the expression of functional EFE 23,24 .…”

The Effect of Promoter and RBS Combination on the Growth and Glycogen Productivity of Sodium-Dependent Bicarbonate Transporter (SbtA) Overexpressing Synechococcus sp. PCC 7002 Cells

“…Cyanobacteria are fast-growing prokaryotic microorganisms capable of oxygenic photosynthesis. They offer advantages of small genome size and genetic amenability (Gantt, 2011;Nozzi et al, 2013;Singh et al, 2016;Jaiswal et al, 2018;Ahmad et al, 2020) and serve as attractive hosts for synthetic biology applications, ranging from metabolic engineering for the production of industrial biochemicals to microbial energy storage (Englund et al, 2016;Vavitsas et al, 2019;Sengupta et al, 2020b). Genetic engineering approaches and tools have been developed for some species (Huang and Lindblad, 2013).…”