Characterization of environmental cellulose-producing isolates of Komagataeibacter xylinus

Abstract

Bacterial cellulose (BC) is a versatile and biocompatible polysaccharide produced by bacteria, with Komagataeibacter xylinus being the model species for its production. This polymer has unique properties that make it valuable in several applications, including as a rheology modifier in cosmetics. Cellugy, a Danish start-up, produces EcoFLEXY, a BC-based rheology modifier that enhances the viscosity and stability of cosmetic formulations while giving a pleasant texture. This product is synthesized by a natural isolate of K. xylinus, strain 7. To enhance the cost-effectiveness and market competitiveness of EcoFLEXY, this thesis aimed to characterize 10 new K. xylinus isolates, comparing their cellulose titer and product quality to strain 7 to identify a more efficient producer. Finally, the study aimed to elucidate the factors contributing to the performance differences among the strains, focusing on strain fitness and Single Amino acid Polymorphisms (SAPs). Among the tested strains, 5 showed a higher titer, with 3 of them producing a high-quality product. Notably, only one strain of these, strain 4, exhibited optimal performance without fitness detriments. Hence, it was chosen as a better strain to use instead of strain 7. Additionally, it was confirmed that differences in production were not due to a variance in strain fitness. When checking SAPs that were present exclusively in strains with a specific phenotype, 10 were selected as relevant to further study. When mapping them on the corresponding enzyme structures, no straightforward conclusions could be identified they were not involved in catalytic reactions, although two were close to the active site. Since the genomic analysis alone did not justify the observed phenotypic differences, it was hypothesized that other factors such as differential gene expression levels were involved. For this reason, further studies including transcriptomic should be performed to elucidate the causes underlying strain performance differences.

Popular Abstract

May the best producer win

Have you ever wondered why the beads in your facial exfoliator do not settle at the bottom? The answer might be cellulose. Cellulose is a natural compound produced by organisms such as plants or bacteria. Due to its structure, this compound adds viscosity to formulations and stabilizes particles. Because of these properties, the company Cellugy decided to produce bacterial cellulose (BC) to use it as an ingredient in cosmetic products. This BC is obtained through a fermentation process using a strain they isolated from natural sources: strain 7, the reference strain.

Strain 7 is an isolate of Komagataeibacter xylinus that produces a high-quality BC in an acceptable quantity. However, although this strain was selected as the best one, Cellugy possesses other strains that have not been characterized. These strains - 10 in total - might have a better performance. Investigating their capabilities was the main aim of this master's thesis. Additionally, the second aim was to determine the causes of performance differences among the strains.

First, the amount of cellulose produced by each strain was quantified. As a result, it was discovered that 5 of the investigated strains produced more than strain 7. This is a significant finding, as producing more would reduce production costs. This would make BC a strong candidate on the market. Therefore, manufacturers could add it to their products instead of other petrochemical-derived ingredients commonly used, making the product more sustainable.

Second, it was verified that the BC produced was of the same quality as that from strain 7. In this case, only 3 strains achieved results equivalent to the reference strain. From these 3 strains, 2 were high producers. Once the strains were classified, their growth was evaluated. The strains chosen to replace strain 7 must grow well, as this would benefit fermentation. Of the 2 previously selected strains, only one grew adequately: strain 4, the champion.

But why were these differences observed? The first hypothesis was that strains were producing more because they grew more, but this was ruled out based on the results of the previous experiment. The second hypothesis was that the strains producing more had something different in them that made them superior: mutations in their genome.

The genome is like a book containing all the information of an organism. It is organized into genes, which are expressed to produce proteins. These proteins perform functions such as producing cellulose. Even if two strains have the same gene, if that gene has a different letter (mutation), the protein coded by it may have different properties. Thus, it was decided to analyze genes related to cellulose production, searching for mutations present only in special strains, such as those producing more. Then, those mutations were located in the enzyme's structure to determine how they might be affecting its function. A mutation in a part with no function is different from one in a part crucial to the enzyme’s activity. Unfortunately, the mutations found were located in areas distant from the important regions, and no conclusive findings could be made. Therefore, further studies to uncover why the strains behave differently are needed. This will enable us to improve the strains we have and reduce fermentation costs even more.

Master’s Degree Project in Molecular Biology 45 credits 2024
Department of Biology, Lund University

Advisor: Antonin Cros and Deby Fapyane
Cellugy

• author: Castiñeira Rial, Nerea
• supervisor: Antonin Cros; Deby Fapyane
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9176112
• date added to LUP: 2024-10-04 13:06:35
• date last changed: 2024-10-04 13:06:35