Methods and products for production of wax esters (u.s. national phase)

a technology of wax esters and products, applied in the field of genetically engineered microorganisms, can solve the problems of increasing demands, reducing production efficiency, and reducing and achieves the effect of high-level production of faeces and increasing the flux of fatty acid biosynthesis

Inactive Publication Date: 2013-08-01
BIOPETROLIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]The above presented problems have now been solved by providing a fungal cell system for producing fatty acyl ethyl esters (FAEE), said system comprising a fungal cell and an expression vector encoding at least one wax synthase, wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosyn

Problems solved by technology

However, fresh discoveries of deposits are on the wane and demands are increasing.
The current process has several drawbacks, including energy intensiveness, consuming edible feedstocks, difficulty of removal of the catalyst from the product and treatment of toxic waste-water, as well as geographical and seasonal restrictions.
The main drawbacks with this kind of enzymatic catalysis are the strong inactivation effect caused by alcohols (i.e. methanol) and the high enzyme costs.
Both chemical and enzymatic transesterification require the use of toxic, petrochemically-derived alcohols and expensive feedstocks.
Thus, transesterification-based biodiesel becomes unsustainable when fossil fuel derived products are used.
However such plant oils are inherently limited by supply of water and land, and subsequently, they cannot produce enough biofuel without threatening food supplies and/or native biodiversity.
Nevertheless, there are problems w

Method used

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  • Methods and products for production of wax esters (u.s. national phase)
  • Methods and products for production of wax esters (u.s. national phase)
  • Methods and products for production of wax esters (u.s. national phase)

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Biodiesel Production Host Saccharomyces cerevisiae CB1

[0128]In this experiment the wax ester synthase from A. baylyi ADP1 was expressed in a laboratory strain Saccharomyces cerevisiae CEN.PK113-5D (MAT-alpha ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2) to create biodiesel producer, Saccharomyces cerevisiae CB1.

[0129]Briefly, cloning and DNA manipulations were all carried out in E .coli DH5α and were performed by standard procedures (Sambrook and Russell 2001). The sequence of the gene atfA with the reported wax synthase from Acinetobacter baylyi ADP1 was optimized for expression in a yeast host. The optimized sequence is given as SEQ ID NO 1, which was based on the published gene sequence (Gene bank accession no. AF529086). It was synthesized and provided by the DNA2.0 Company (Menlo Park, Calif.). SEQ ID NO 1 was amplified using the following oligonucleotides: 5′-CGGGATCCCGCTCGAGATGCGTCCATT-3′ (SEQ ID NO 2) introducing BamHI restriction site (underlined) and; 5′-GGGGTACCCCA...

example 2

Characteristics of the Recombinant Host

[0130]The inoculated transformants S. cerevisiae CB0 and S. cerevisiae CB1 were cultured to late exponential growth period in 100 mL SD medium lacking uracil and containing 2% (w / v) glucose at 30° C. The cultures were then harvested. Cell-free extracts were prepared using a previously reported fast prep method for enzyme analysis (Hou, Vemuri et al. 2009). The lipid analysis were extracted from the lyophilized cell pellets using the reported method (Gu, Valianpour et al. 2004).

[0131]The wax synthase activities in the transformants were testified in vitro using [1-14C] palmitoyl-CoA and 1-hexadecanol or ethanol as the substrates. Table 1 summarizes the results of enzyme analysis. A low wax synthase activity could be detected in negative control S. cerevisiae CB0 using 1-hexadecanol or ethanol as the substrates. In contrast, a significant high wax synthase activity was detected in S. cerevisiae CB1.

[0132]The lipid extraction was analyzed with Gas...

example 3

Evaluation of the Substrate Preference of Different WSs in Yeast

[0133]

TABLE 2Specific oligonucleotides used for PCRamplification of the synthesized WS sequencesPrimer Sequence 5′→3′UpstreamDownstreamWS fromCGGGATCCCGCTCGGGGTACCCCAAGCTTGMarinobacterGAGATGAAGAGATTGGTTACTTTCTAGTACGhydrocarbo-AGG(SEQ ID NO 9)noclasticus(SEQ ID NO 8)DSM 8798WS fromCGGGATCCCGCTCGGGGTACCCCAAGCTTGRhodococcusGAGTTGACCGACGGGTTAGCTAGCCACCACCopacus PD630TGATTAC(SEQ ID NO 11)(SEQ ID NO 10)WS from MusCGGGATCCCGCTCGGGGTACCCCAAGCTTGmusculusGAGATGTTCTGGCCGGTTAAACAATGACCAACC57BL / 6AACC(SEQ ID NO 13)(SEQ ID NO 12)WS fromCGGGATCCCGCTCGGGGTACCCCAAGCTTPsychrobacterGAGATGAGATTACTGGGTTAAGGGGCCAACTarticus 273-4GACCGCTGT(SEQ ID NO 15)(SEQ ID NO 14)

[0134]In this example, except for the wax synthase from Acinetobacter baylyi ADP1, four other putative WSs from Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL / 6, and Psychrobacter articus 273-4 were optimized for expression in a yeast host....

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Abstract

The present invention relates to the provision of genetically modified fungal cells, such as yeast cells with an improved ability for producing different fatty acids and specifically fatty acid ethyl esters (FAEE), the main components of biodiesel. An increased in fatty acid production, and hence in FAEE, is obtained in the first place by expressing different heterologous polypeptides in combination with the down-regulation, attenuation, deletion or over-expression of specially selected genes, wherein said genes encode enzymes involved in the fatty acids synthesizing pathway, fatty acid consuming pathways, carbohydrate biosynthesis pathways or enzyme acting as wax ester transporters or a combination thereof. The methods and products of the invention would allow large-scale production of FAEE with carbohydrates as the only externally-supplied substrate.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to the development of genetically engineered microorganisms that can produce wax esters in a controllable and economic fashion. More specifically the invention relates to the production of liquid wax esters that can be used for biofuel, lubricants, cosmetics, linoleum, printing inks as well as products related thereto, and for the production of solid wax esters used for candles and polishes as well as products related thereto.[0003]2. Description of the Related Art[0004]Fossil fuels, such as coal, oil, and natural gas, have been powering modern society for more than one century. However, fresh discoveries of deposits are on the wane and demands are increasing. The world's demand of fossil fuels will soon outweigh the current supply. An innovative approach offering some solution comes from the biotechnology industries. Efforts have made biodiesel as one of the most thoroughly developed and p...

Claims

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Application Information

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IPC IPC(8): C12P7/64
CPCC12N9/1029Y02E50/13C12P7/649C12N15/81Y02E50/10C12Y602/01001
Inventor NIELSEN, JENSSHI, SHUOBO
Owner BIOPETROLIA
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