Browsing by Subject "Pseudomonas putida KT2440"

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Biotechnological conversion of lignocellulose hydrolyzates : model microorganisms for a bio-based economy
(2020) Horlamus, Felix; Hausmann, Rudolf
Lignocellulose has substantial potential as a carbon source in a bio-based economy. It is the most abundant renewable raw material on earth and is available in large quantities as waste from the agriculture, food and wood industry. It is composed mainly of the polymers lignin, cellulose and hemicellulose. In contrast to glucose derived from cellulose, hemicellulose sugars often remain unused although 60 billion tons of hemicelluloses are produced annually. Hemicelluloses are a group of heterogeneous polysaccharides consisting of different monomers such as D xylose, D arabinose, D mannose and D galactose. Lignocellulose is mostly depolymerized in order to obtain fermentable sugars. During the depolymerization process, inhibitors such as organic acids or furan aldehydes can be formed or released, which could be problematical for biotechnological processes. The aim of this thesis was to develop and evaluate bacterial-based biotechnological processes capable of using hemicellulose sugars as a source of carbon. First, Pseudomonas putida KT2440 was chosen. Pseudomonades are claimed as a promising chassis in biotechnology due to their versatile and robust metabolism. Unlike other Pseudomonades, the strain KT2440 is classified as biosafety level 1 in the American Type Culture Collection (ATCC). However, these bacteria can metabolize glucose as the only lignocellulose monosaccharide. Cellvibrio japonicus was the second selected bacterium. This strain is not yet established as a microbial host in biotechnology, but can degrade a huge portfolio of plant cell wall polysaccharides and is also classified as biosafety level 1 in ATCC. The topic of the first publication was to engineer P. putida KT2440 strains for metabolizing the hemicellulose monosaccharides xylose and arabinose and characterize their growth behavior. Initially, an arabinose metabolizing strain with the araBAD operon and a xylose metabolizing strain with xylAB operon was constructed. Later on, these strains were cultivated in minimal salt medium with glucose, xylose and arabinose as carbon sources in Erlenmeyer flasks. The recombinant P. putida KT2440 strains metabolized xylose and arabinose with high growth rates comparable to glucose. It turned out that both engineered strains were able to grow on both pentoses as well as on mixtures of glucose xylose and arabinose. The intent of the second publication was to evaluate P. putida KT2440 as a platform model organism for bioconversion of lignocellulose hydrolyzates. Strains were cultivated in minimal salt medium with several hydrolyzates as carbon source in Erlenmeyer flask and bioreactor. In addition, the growth-inhibiting effect of major toxic substances contained in lignocellulose hydrolyzates on P. putida KT2440 was analyzed via cultivation experiments. Several suitable hydrolyzates were figured out for this strain. Formic acid and acetic acid proved to be relatively unproblematic under pH neutral conditions, whereas furfural and hydroxymethylfurfural (HMF) had a negative effect on the bacterial growth. A diauxic-like growth behavior was revealed via fed batch bioreactor cultivations, since pentoses were almost not consumed with sufficient glucose supply. Consequently, feed-medium was added step-by-step in the next experiment. The applied feed profile did lead to an almost complete metabolization of xylose. The purpose of the third publication was to evaluate C. japonicus as a potential host strain for the one‐step bioconversion of xylans into rhamnolipids. Cultivation experiments were performed in Erlenmeyer flasks filled with minimal salt medium and containing different carbon sources. Furthermore, the strain was transformed with the plasmid pSynPro8oT carrying rhlA (encodes acetyltransferase) and rhlB (encodes rhamnosyltransferase I) to complete the rhamnolipid metabolism. The strain grew on all main lignocellulose monosaccharides as well as, on different xylans. Mono rhamnolipids were produced with the engineered strain using xylans as carbon source. This is particularly interesting as most industrially relevant bacteria are not able to depolymerize wood polymers. As the product yields were quite low, there are still many challenges in order to achieve an economically efficient process. Nevertheless, to the best of our knowledge, it is the first published one step bioconversion of hemicellulose polymers into rhamnolipids. In total, P. putida KT2440 turned out as a flexible and powerful model organism and two xylose and arabinose metabolizing strains were constructed. Moreover, bioreactor cultivations with lignocellulose hydrolyzates were performed and a feeding strategy to overcome diauxic-like growth behavior was presented. A proof of concept for a one-step bioconversion of xylans into rhamnolipids with a recombinant C. japonicus strain was successfully demonstrated.