Engineering of Chromosomal Wax Ester Synthase Integrated Saccharomyces Cerevisiae Mutants for Improved Biosynthesis of Fatty Acid Ethyl Esters
Journal article, 2014

In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

delta sequence

homologous recombination

metabolic engineering

gapN

FAEEs

ACBP

Author

Shuobo Shi

Chalmers, Chemical and Biological Engineering, Life Sciences, System Biology

Juan Octavio Valle Rodriguez

Chalmers, Chemical and Biological Engineering, Life Sciences, System Biology

Verena Siewers

Chalmers, Chemical and Biological Engineering, Life Sciences, System Biology

Jens B Nielsen

Chalmers, Chemical and Biological Engineering, Life Sciences, System Biology

Biotechnology and Bioengineering

0006-3592 (ISSN) 1097-0290 (eISSN)

Vol. 111 9 1740-1747

Areas of Advance

Life Science Engineering (2010-2018)

Subject Categories

Bioinformatics and Systems Biology

DOI

10.1002/bit.25234

More information

Created

10/7/2017