Alkane biosynthesis by Aspergillus carbonarius ITEM 5010 through heterologous expression of Synechococcus elongatus acyl-ACP/CoA reductase and aldehyde deformylating oxygenase genes

Malavika Sinha,Kenneth S Bruno,István Weyda,Birgitte K Ahring,Annette Sørensen

doi:10.1186/s13568-016-0321-x

Malavika Sinha, Kenneth S Bruno + Show 3 more

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https://doi.org/10.1186/s13568-016-0321-x

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Abstract

In this study we describe the heterologous expression of the recently identified cyanobacterial pathway for long chain alkane biosynthesis, involving the reduction of fatty acyl-ACP to fatty aldehyde and the subsequent conversion of this into alkanes, in the filamentous fungus Aspergillus carbonarius ITEM 5010. Genes originating from Synechococcus elongatus strain PCC7942, encoding acyl-ACP/CoA reductase and aldehyde deformylating oxygenase enzymes, were successfully expressed in A. carbonarius, which lead to the production of pentadecane and heptadecane, alkanes that have not been previously produced by this fungus. Titers of 0.2, 0.5 and 2.7 mg/l pentadecane and 0.8, 1.6 and 10.2 mg/l heptadecane were achieved using glucose, Yeast malt and oatmeal media, respectively. Besides producing alkanes, we found elevated levels of internal free fatty acids and triglycerides in the alkane producing transformant. These findings can indicate that a yet unidentified, native fatty aldehyde dehydrogenase channels back the fatty aldehydes into the fatty acid metabolism, thus competing for substrate with the heterologously expressed fatty aldehyde deformylating oxygenase. These findings will potentially facilitate the future application of robust, fungal cell factories for the production of advanced biofuels from various substrates.

Highlights

Microbial production of fatty acid derived biofuels, such as fatty acids, fatty alcohols and alkanes has received considerable interest in the past few years as a mean for producing advanced biofuels which can substitute conventional transportation fuels (Fairley 2011)
Selection of correct transformants and southern blot analysis The transformants were verified by PCR to determine if integration of the vector construct had occurred, followed by analysis of expression from Complementary DNA (cDNA) (Fig. 2).The quality of the synthesized cDNA of the transformants was verified by amplifying a short terminal fragment of the beta- actin gene as control
The amounts of pentadecane produced from glucose, yeast malt (YM) and oatmeal media were 0.2, 0.5 and Discussion The heterologous expression of the S. elongatus PCC7942 codon optimized FAR and FADO in A. carbonarius ITEM 5010 resulted in the production of alkanes, pentadecane and heptadecane, which have not previously been seen produced by this fungus (Sinha et al 2015)

Summary

Introduction

Microbial production of fatty acid derived biofuels, such as fatty acids, fatty alcohols and alkanes has received considerable interest in the past few years as a mean for producing advanced biofuels (or drop-in fuels) which can substitute conventional transportation fuels (Fairley 2011). Aldehydes into Cn−1 alkanes, predominantly pentadecane, heptadecane and methyl-heptadecane, in various cyanobacteria In this same study, the heterologous expression of these genes in E. coli led to alkane titers of 25 mg/l, and based on these findings significant progress has further been made with this microorganism, achieving alkane titers of up to 580 mg/l (Choi and Lee 2013). One study on Saccharomyces cerevisiae (Buijs et al 2015) reported, that the successful expression of FAR and FADO together with the deletion of a fatty aldehyde dehydrogenase (FALDH), encoded by Hfd, led to the production of long chain alkanes in titers of 22 μg/g dry cells mass. The Hfd enzyme is related to accumulation of fatty acids by degrading fatty aldehydes that would otherwise serve as substrate for the reaction carried out by the FADO enzyme. The production of fatty aldehydes in cells is related to the catabolism of several lipids, such as fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols, etc. (Rizzo 2014)

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