Producing aliphatic hydrocarbon biofuels using microorganisms has been an attractive idea since the discovery of the natural alkane-producing species and various alkane synthesis pathways. Though many efforts have been made to improve the hydrocarbon productivities of the recombinant microbial cell factories, the yields are abysmally low, with a huge distance to the further industrial applications.  

Recently, Prof. LU Xuefeng and his team from Microbial Metabolic Engineering Group, Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT), Chinese Academy of Sciences, have started a research project on cyanobacterial hydrocarbon production, which is partly focusing on the engineering and evolution of the enzyme aldehyde deformylating oxygenase (ADO). As the last enzyme of acyl-ACP reductase (AAR)-ADO alka(e)ne synthesis pathway, ADO catalyzes the transformation of fatty aldehydes into fatty alkanes. However, the enzyme has very poor catalytic efficiency and stability, and thus is one of the bottlenecks in the alkane biosynthesis. 

One of the key challenges to preform evolution is how to find out the particular ADO mutants with higher catalytic efficiency. To solve this problem, as reported in a newly published work (online in Scientific Reports on June 3^rd, 2015), the postdoctoral researcher Dr. WU Wei and PhD student ZHANG Lei from the team have just developed a convenient fluorescent biosensor that is able to monitor the alkane synthesis in Escherichia coli, which is a commonly used host for ADO evolution. The approach was achieved by integrating a genetically recombinant bacterial alkane biosensor into the expressing hosts (e.g. E. coli) which already harbored the AAR-ADO pathway. Generally, the biosensor mainly consisted of three DNA segments: one encoding an alkane responsive transcriptional regulator AlkR, the AlkR-controlled promoter P_alkM, and one encoding green fluorescent protein (GFP) as reporter protein. Alkane synthesized under isopropyl β-D-1-thiogalactopyranoside (IPTG) induction could be detected by the biosensor, which then initiated the GFP expression. In this way, the alkane concentration signal could be converted into the concentration-dependent fluorescence signal which could be easily detected using a fluorescence microplate reader or a flow cytometer.  

It is for the first time that a simple and convenient biosensor has been constructed for monitoring of the mid- and long-chain alkane synthesis in E. coli, suggesting a high-throughput approach for the quick evaluation of the alkane synthesis efficiency for different ADO mutants, even in a single cell level.  

This work is funded by National Basic Research Program of China (973), National Natural Science Foundation of China, China Postdoctoral Science Foundation and the Joint Research Laboratory for Sustainable Aviation Biofuels (Boeing-QIBEBT). 

Figure 1 (a) Schematic for the in situ probing of endogenous alkane in E. coli basing on the synthetic alkane biosensor cARE. (b, c, d) Dynamic cARE response of the strains harboring the biosensor. (Image by QIBEBT)