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New processes for the fermentative production of glycolipid biosurfactants and sialylated carbohydrates

Periodic Reporting for period 2 - CARBOSURF (New processes for the fermentative production of glycolipid biosurfactants and sialylated carbohydrates)

Reporting period: 2017-02-01 to 2018-07-31

In the Carbosurf project, the hurdles that hamper the full exploitation of two specialty biochemical classes with high market interest were addressed, namely glycolipid biosurfactants (new types of sophorolipids and sophorosides, rhamnolipids, mannosylerythritol lipids (MELs) and xylolipids) and specialty carbohydrates (sialyllactose and LactoNtetraoses). Existing technology platforms were challenged to meet specific market needs. Tailor-made molecules with promising market potential were developed in close cooperation with relevant major market players. This ensured that scientific developments followed a market-driven approach and guaranteed that maximum results for the technology platforms were achieved.

The overall objectives of the Carbosurf project were:
- Improved metabolic engineering and synthetic biology methodologies to shorten development times and time to market
- Availability of robust production strains to obtain well-defined molecules
- A competitive production system able to compete with currently used (petrochemically-based) alternatives
- Prove of efficacy in commercial applications

All milestones were achieved, and the majority of the objectives were reached. Thus, the CarboSurf project was successful. Most of the Carbosurf biochemicals will soon enter the market, which will have several beneficial impacts on society.
The conversion of agricultural waste products (i.e. lignocellulosic material) into fermentable sugars was optimized, which resulted in higher purity and a lower price of these second generation (2G) sugars. It was shown that these 2G sugars can efficiently be fermentatively converted into biosurfactants (sophorolipids, xylolipids and MELs), which improves the environmental profile of the latter as shown in an LCA comparing the environmental impact of first generation (1G) sophorolipids to 2G sophorolipids.

New molecular tools were developed for a range of microbial strains included in the CarboSurf project (bacterial, yeasts and fungi). These tools significantly facilitate(d) the metabolic engineering of several glycolipid and carbohydrate producing strains. A set of microbial strains efficiently producing (new types) of glycolipids (new-to-nature sophorolipids and sophorosides, rhamnolipids and xylolipids) and producing specialty carbohydrates were thus created.

The described (new-to-nature) biomolecules hold great intrinsic potential for commercialization in a range of sectors such as food, cosmetics, personal care, detergents, nutraceuticals, etc. To assess their potential, a set of physicochemical and biological parameters were investigated and for some molecules, specific application tests were performed by the industrial partners of the CarboSurf project. For some very promising results were obtained e.g. xylolipids as biological preservatives in beverages and rhamnolipids and bola sophorosides as green and mild performers in personal care formulations. To perform these application tests, considerable quantities of the products of high purity were required and to reach these goals, suitable and scalable production processes for the new strains/products had to be developed. For the new types of sophorolipids and sophorosides, the processes for promising strains were developed at the lab scale and scaled up to the 150 L scale, giving range to several 100 grams of the new product(s) ((acetylated) symmetrical bola sophorosides and (acetylated) alkyl sophorosides). A lot of attention was given to purification development as this is often underestimated, but a very important aspect towards commercialization. For the rhamnolipids, the production process was debottlenecked at the ton scale (1500 L) and possible commercial scale issues (100 ton scale) were identified and resolved. Also, for xylolipids, pilot scale (150 L) production runs were performed after developing a suitable fermentation and purification process at the lab scale. For the fourth type of glycolipid (mannosylerythritol lipids (MELs)), smaller scale fermentations were performed to investigate the purification and produce product for application tests. For the specialty carbohydrates product line, we have developed production hosts and processes for specific sialylated oligosaccharides and have identified novel applications for these oligosaccharides.
The most important progress point beyond the state of the art of the CarboSurf project are:

- Availability of a new process for the efficient and economical production of 2G sugars.
- Production of biosurfactants from the optimized 2G sugars, dramatically improving their environmental profile.
- New molecular tools or methods available for Starmerella bombicola, Saccharomyces cerevisiae, Escherichia coli and Pseudozyma strains.
- Availability of microbial strains efficiently producing (new-to-nature) sophorolipids and sophorosides, rhamnolipids, xylolipids and specialty carbohydrates.
- New production processes for the new strains/biomolecules ((acetylated) symmetrical bola sophorolipids, alkyl sophorosides, rhamnolipids, xylolipids, MELs, sialylated oligosaccharides).
- Application data available for new biochemicals.

The results obtained so far point towards the market entry of several new biochemicals in Europe, which has several beneficial impacts on society. Firstly, moving these innovative products to the market will boost economic development and employment in Europe. Secondly, as the biochemicals are biodegradable and non-ecotoxic and are being produced from (second generation) renewable resources, this will have a positive effect on the environment. Thirdly, a positive social impact is expected as (some of) the biochemicals have high potential in pharmaceutical and nutraceutical applications and as antibiotic alternatives, all improving human quality of life.