Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12mol% 3HV from 0.5g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500L pilot-scale fermentor studies lasting 70h, the above engineered Halomonas TD01 grew to 112g/L CDW containing 70wt% P3HB, and to 80g/L CDW with 70wt% P(3HB-co-8mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.
Halophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far.
The production of 2, 3-butanediol and succinic acid by a moderate halophile under anaerobic condition was investigated. This halophile, termed Salinivibrio YS, was isolated from the solid samples collected from Aydingkol Lake. Based on the single factor experiment, the parameters and their values for the production were obtained. Then, the optimum values of these parameters by the orthogonal experiments were obtained: temperature, 33 degrees C; initial pH of fermentation, 8.0; the pH during fermentation, 7.0; the concentration of acetic acid was 3 g/L and NaC1 was 10 g/L. Finally, a 3-L fermentation based on these conditions was carried out. After 108 h of fermentation under anaerobic condition, 35.05 g/L of 2, 3-butanediol and 22.46 g/L of succinic acid were obtained. About 50% of total glucose conversion was achieved. The study on 2, 3-butanediol and succinic acid by a halophile under anaerobic condition will expand the applications of halophiles and open a new area of production of 2, 3-butanediol and succinic acid.
An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.