Enzymes for consumer products to achieve climate neutrality

29 pags., 4 figs., 3 tabs., 1 graf.Accumulated greenhouse gas emissions are expected to increase from 36.2 Giga-tons (Gt) to 60 Gt over the next three decades. The global surface temperature has increased by¿+¿1.09¿°C since 2001, and might increase by¿+¿2.2¿°C in 2100, +3.6¿°C in 2200 and +4.6¿°C in 2500. These emissions and temperature rises cannot be reduced in their entirety, but they can be lowered by using enzymes. Enzymes are proteins that catalyze biochemical reactions that make life possible since 3.8 billion years ago. Scientists have been able to "domesticate" them in such a way that enzymes, and their engineered variants, are now key players of the circular economy. With a world production of 117 Kilo-tons and a trade of 14.5 Billion-dollars, they have the potential to annually decrease CO2 emissions by 1 to 2.5 Billion-tons (Bt), the carbon demand to synthesise chemicals by 200 Million tons (Mt), the amount of chemicals by 90¿Mt, and the economic losses derived from global warming by 0.5%, while promoting biodiversity and our planet¿s health. Our success to increase these benefits will depend on better integration of enzymatic solutions in different sectors.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. MF also acknowledges Grants PID2020-112758RB-I00, PDC2021-121534-I00, and TED2021-130544B-I00 from the MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”)

Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides ▿

Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains