RESULTS OF PROJECT 4, December 2018

The goal of project 4 was to develop and demonstrate the microbial production of the amino acid lysine from C5 and C6 sugar streams based on lignocellulosic feedstocks to be supplied by project 3.

Compared with the traditional microbial technology platform which has been used for many decades for the production of lysine from sugars such as glucose and sucrose, using lignocellulosic feedstocks pose at least two distinct problems which had to be solved by project 4:

The first problem was that during the conversion of lignocellulose into sugar hydrolysates, many chemicals such as furfural, vanillin, HMF, etc. will be formed which are known to inhibit the subsequent microbial fermentation process based on isolates of Corynebacterium glutamicum. In project 4 we had two strategies to circumvent this problem: either we could try to adapt the lysine producing strains to the new growth conditions in lignocellulosic hydrolysates or we could start all over from another bacterial platform based on lactid acid bacteria which are well known to have high tolerance to the above-mentioned chemicals and then convert one of these strains into a lysine producer. The first strategy was proven to be successful and during the course of the project we managed to adapt C. glutamicum by serial transfer in medium with inhibitors to grow in the presence of relatively high concentrations of lignocellulosic hydrolysate and the accompanying inhibitors. Our conclusion was therefore that Corynebacterium glutamicum was a suitable platform for lysine production also when lignocellulose hydrolysates are used as the sugar source.

The second problem we faced was the fact that Corynebacterium glutamicum is well known to be unable to ferment xylose, the C5 carbon sugar which is present in the lignocellulose hydrolysates. The inability to grow on and ferment xylose would make the process non-competitive in an industrial setup since xylose is a major part of the sugars. In the project we first engineered a model strain of C. glutamicum to an efficient lysine producer by introducing several mutations in the bacterial genome. In the process we also discovered several interesting mechanisms which are involved in lysine metabolism and showed that changing some of these can improve the lysine production on certain sugar sources. Subsequently, in order to make C. glutamicum able to grow on xylose, we introduced a set of xylose transporting and metabolising genes on a plasmid which resulted in a xylose fermenting mutant of C. glutamicum which was capable of efficiently producing lysine from both xylose and glucose. Interestingly, our results revealed that in contrast to our expectations, xylose appeared to be a more efficient substrate for lysine production compared to glucose.

Finally, towards the end of the project, we also managed to transfer the constructed plasmid to the industrial production strain, which was used for many years by the company Vitalys for lysine production, and successfully managed to produce lysine from both glucose and xylose also with this production strain.

In combination with the results described above on improved inhibitor tolerance, our data suggests that cheap xylose or mixed fractions of lignocellosic hydrolysates could potentially be attractive as future feedstocks for a more sustainable production of lysine and other food/feed ingredients.