BackgroundMicrobial bioconversion of photosynthetic biomass is a promising approach to the generation of biofuels and other bioproducts. However, rapid, high-yield, and simple processes are essential for successful applications. Here, biomass from the rapidly growing photosynthetic marine cyanobacterium Synechococcus sp. PCC 7002 was fermented using yeast into bioethanol.ResultsThe cyanobacterium accumulated a total carbohydrate content of about 60% of cell dry weight when cultivated under nitrate limitation. The cyanobacterial cells were harvested by centrifugation and subjected to enzymatic hydrolysis using lysozyme and two alpha-glucanases. This enzymatic hydrolysate was fermented into ethanol by Saccharomyces cerevisiae without further treatment. All enzyme treatments and fermentations were carried out in the residual growth medium of the cyanobacteria with the only modification being that pH was adjusted to the optimal value. The highest ethanol yield and concentration obtained was 0.27 g ethanol per g cell dry weight and 30 g ethanol L-1, respectively. About 90% of the glucose in the biomass was converted to ethanol. The cyanobacterial hydrolysate was rapidly fermented (up to 20 g ethanol L-1 day-1) even in the absence of any other nutrient additions to the fermentation medium.ConclusionsCyanobacterial biomass was hydrolyzed using a simple enzymatic treatment and fermented into ethanol more rapidly and to higher concentrations than previously reported for similar approaches using cyanobacteria or microalgae. Importantly, as well as fermentable carbohydrates, the cyanobacterial hydrolysate contained additional nutrients that promoted fermentation. This hydrolysate is therefore a promising substitute for the relatively expensive nutrient additives (such as yeast extract) commonly used for Saccharomyces fermentations.

Background Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO 2 , light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant Tf AA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Results Two variations of Tf AA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine- N -oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA- Tf AA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of Tf AA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. Tf AA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L −1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. Conclusions We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis. Electronic supplementary material The online version of this article (10.1186/s13068-019-1416-9) contains supplementary material, which is available to authorized users.

Lytic polysaccharide monooxygenases (LPMOs)a re copper-containing enzymes capable of oxidizing crystalline cellulose whichh ave large practical application in the process of refiningb iomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report al ong-lived intermediate( t1 = 2 = 6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with as trong absorption around 420 nm is formed when reduced LPMO-Cu I reacts with sub-equimolar amounts of H 2 O 2 .U V/Vis absorption spectroscopy,e lectron paramagnetic resonance,r eso-nanceR amana nd stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper centera nd an earbyt yrosine (Tyr175). Additionally,a ctivity assaysi nt he presence of sub-equimolar amountso fH 2 O 2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly,t his suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. Theo bserved intermediate offers an ew perspective into the oxidative reaction mechanism of Ta LPMO9A and hence for the biomass oxidation and the reactivityo fc opperi nb iologicals ystems.[**] Ap revious versiono ft his manuscript has been deposited on ap reprint server (https://doi.org/10.1101/610865).Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.