Cyanobacteria, the earliest prokaryotes that perform oxygenic photosynthesis on the earth, can utilize sunlight, water, carbon dioxide and inorganic nutrients to synthesize saccharides, proteins, lipids and other organics. Wax esters found in plants, animals as well as microorganisms are oxoesters formed from long-chain fatty acids and long-chain fatty alcohols. Wax esters can be used as raw materials to manufacture lubricants used in accurate instruments, additives to medicine, foods or cosmetics, therefore, have high commercial values. However, natural wax esters are of limited resources and relatively expensive. The first part of my thesis describes the introduction of a bacterial and a mammalian gene, both involved in wax ester synthesis, into Anabaena sp. and a derivative to obtain transgenic strains capable of wax ester synthesis, proposing a new and more cost-saving route for large scale production of wax esters. Diesel, as an important product of oil refining, takes a high position in the world’s fuels markets. Due to the increasingly rapid exhaustion of the earth’s fossil energy resources, many contries are exploring alternative energy sources. For its extrodinary features in environment protection, biodiesel is attracting more and more interst. The second part of my thesis describes the introduction of two bacterial acohol synthesis genes and a wax ester synthesis gene into Escherichia coli to construct a recombinant strain that produces ethyl biodiesel.
Experimentation and results are summarized as follows:
1. The mouse FAR1 cDNA encoding a bifunctional acyl-coenzymeA reductase (FAR1) was cloned from Mus musculus strain KM by RT-PCR. The FAR1 gene and wax/dgat encoding wax ester synthase (WS/DGAT), cloned from Acinetobacter baylyi strain ADP1, were both positioned under the control of PrbcL, resulting in an expressive shuttle vector. The shulte vector was introduced into Anabaena sp. PCC 7120 (referred to as WT in this thesis) and a derivative EF113 by conjugation, resulting in transgenic strains showing wax ester synthesis. The strain EF113 was more productive in wax esters than the WT, amounting to ca.1% cell dry weight.
2. pdc encoding pyruvate decarboxylase (PDC) and adhB encoding alcohol dehydrogenase (ADHB) were cloned from Zymomonas mobilis. An expressive plasmid was constructed and used to coexpress the two genes and wax/dgat althogether from the promoter PlacZ in E.coli DH5α. In the presence of exogenous glucose and sodium oleate, the recombinant strain was able to accumulate fatty acid ethyl esters.