Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae

Abstract

Background: Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemical production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bottleneck for alkane biosynthesis in Saccharomyces cerevisiae. We therefore performed functional screening to identify efficient ADs that can improve alkane production by S. cerevisiae.

Results: A comparative study of ADs originated from a plant, insects, and cyanobacteria were conducted in S. cerevisiae. As a result, expression of aldehyde deformylating oxygenases (ADOs), which are cyanobacterial ADs, from Synechococcus elongatus and Crocosphaera watsonii converted fatty aldehydes to corresponding C_n-1 alkanes and alkenes. The CwADO showed the highest alkane titer (0.13 mg/L/OD₆₀₀) and the lowest fatty alcohol production (0.55 mg/L/OD₆₀₀). However, no measurable alkanes and alkenes were detected in other AD expressed yeast strains. Dynamic expression of SeADO and CwADO under GAL promoters increased alkane production to 0.20 mg/L/OD₆₀₀ and no fatty alcohols, with even number chain lengths from C8 to C14, were detected in the cells.

Conclusions: We demonstrated in vivo enzyme activities of ADs by displaying profiles of alkanes and fatty alcohols in S. cerevisiae. Among the AD enzymes evaluated, cyanobacteria ADOs were found to be suitable for alkane biosynthesis in S. cerevisiae. This work will be helpful to decide an AD candidate for alkane biosynthesis in S. cerevisiae and it will provide useful information for further investigation of AD enzymes with improved activities.

Keywords: Aldehyde decarbonylase; Alkane biosynthesis; Biofuels; Metabolic engineering; Saccharomyces cerevisiae.

Fig. 1

Scheme of alkane biosynthesis in engineered S. cerevisiae strains. The genes encoding fatty acyl-CoA oxidase, POX1, aldehyde dehydrogenase, HFD1 and alcohol dehydrogenase, ADH5, were disrupted (blue) and alcohol dehydrogenase was overexpressed (red). ADs were inserted in an episomal plasmid and they were expressed to convert fatty aldehydes to alkanes (green)

Fig. 2

Comparison of alkane and fatty alcohol production by different AD expression in engineered S. cerevisiae strains. Alkane (a) and fatty alcohol (b) titers, and cell growth (c) were demonstrated from each engineered strain after 72 h culture in minimal media. All data represent the mean values and standard deviations from at least triplicate cultures

Fig. 3

Enhancement of alkane production. Production of alkanes (a) and fatty alcohols (b), and two titer units (left blue mg/L/OD₆₀₀, right orange mg/L) are used to display the level of metabolites. Cell growth of each strain is shown in (c). All data represent the mean values and standard deviations from at least triplicate cultures