Peiru, S. | bioRxiv Science

bioRxiv2022

A highly stable Zn(II) dependent phospholipase C obtained by consensus design

Proteins extraordinary performance in recognition and catalysis have led their use in a range of applications. But proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here we achieved a substantial thermal stabilization of a bacterial Zn(II) dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologs from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of activity and stability parameters of this stabilized mutant and other natural variants bearing similar mutations allow us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in the harsh oil degumming conditions, making it suitable for industrial applications.

biochemistry↗

bioRxiv2021

Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain

Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption. Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed. A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100 %. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP free platform that could be adaptable for production of other biomolecules. Key PointsO_LIA platform for the production of biomolecules was designed based on B. subtilis 3NA, a chemically defined medium and a fermentation process. C_LIO_LIAs proof of concept, high quality hyaluronic acid was produced with an environmentally friendly process. C_LIO_LIA techno-economic analysis indicates that the process is more that 100% profitable than current methods. C_LI

bioengineering↗