生物化学

Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane. However, the variation in substrate availability to the first enzyme of the pathway, i.e., AAR, via fatty acid synthesis pathway and low turnover of the ADO enzyme make calculation of yields and titers under in vivo conditions extremely difficult. In vivo assay employing external addition of defined substrates for ADO enzyme into the medium helps to monitor the influx of substrate hence providing a more accurate measurement of the product yields. In this protocol, we include a detailed guide for implementing the in vivo assay for monitoring alkane production in E. coli.

Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO₂. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures.

Many species of cyanobacteria accumulate alkanes in their cells. It has been reported that these cyanobacteria accumulate mainly 15 long carbon chain alkane, pentadecane (C₁₅H₃₂), or/and 17 long chain alkane, heptadecane (C₁₇H₃₆). Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular pentadecane and heptadecane. We have confirmed this protocol was applicable to at least three kinds of cyanobacteria, nitrogen-fixing filamentous cyanobacterium Anabaena sp. PCC7120, non-diazotrophic unicellular cyanobacterium Synechococcus elongatus PCC7942 and halotolerant unicellular cyanobacterium Aphanothece halophytica.