Abstract
BackgroundLow 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.ResultsA 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 Cn−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.ConclusionsWe 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.
Highlights
Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemi‐ cal production
We introduced three different types of aldehyde decarbonylase (AD), the ECERIFERUM1 (CER1) from Arabidopsis plant [16, 21], insect cytochrome p450s (CYP4G1 and CYP4G2) from D. melanogaster and house fly [12], and cyanobacteria aldehyde deformylating oxidases (ADOs) from S. elongatus [17, 18], Crocosphaera watsonii, Thermosynechococcus elongatus, and Cyanothece sp
Codon-optimized aldehyde deformylating oxygenase (ADO) and CER1 genes were expressed under the control of Plasmids pYX212 pAlkane78 pAlkane8 pAlkane71 pAlkane67 pAlkane83 pAlkane84 pAlkane85 pAlkane86
Summary
Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemi‐ cal production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bot‐ tleneck for alkane biosynthesis in Saccharomyces cerevisiae. Global warming and depletion of fossil fuels are two urgent matters. Fossil fuels are finite energy resources, but the world energy demand has been increasing along with economic development and population growth. There has been growing interest in sustainable production of biofuels and bio-based chemicals using microorganisms, so called cell factories. One of the most important microbial cell factories, Saccharomyces cerevisiae is generally recognized as safe (GRAS) and, it is an extremely well-characterized and tractable organism. Because of its robustness and tolerance towards various stress conditions, it has been intensively used to produce several advanced biofuels and chemicals [6,7,8,9]
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