BackgroundBiosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based alkanes. Acyl-[acyl carrier protein (ACP)] reductase (AAR) is a key enzyme for alkane biosynthesis in cyanobacteria and catalyzes the reduction of fatty acyl-ACP to fatty aldehydes, which are then converted into alkanes/alkenes by aldehyde-deformylating oxygenase (ADO). The amino acid sequences of AARs vary among cyanobacteria. However, their differences in catalytic activity, substrate specificity, and solubility are poorly understood.ResultsWe compared the aldehyde-producing activity, substrate specificity, and solubility of AARs from 12 representative cyanobacteria. The activity is the highest for AAR from Synechococcus elongatus PCC 7942, followed by AAR from Prochlorococcus marinus MIT 9313. On the other hand, protein solubility is high for AARs from PCC 7942, Microcystis aeruginosa, Thermosynechococcus elongatus BP-1, Synechococcus sp. RS9917, and Synechococcus sp. CB0205. As a consequence, the amount of alkanes/alkenes produced in Escherichia coli coexpressing AAR and ADO is the highest for AAR from PCC 7942, followed by AARs from BP-1 and MIT 9313. Strikingly, AARs from marine and freshwater cyanobacteria tend to have higher specificity toward the substrates with 16 and 18 carbons in the fatty acyl chain, respectively, suggesting that the substrate specificity of AARs correlates with the type of habitat of host cyanobacteria. Furthermore, mutational analysis identified several residues responsible for the high activity of AAR.ConclusionsWe found that the activity, substrate specificity, and solubility are diverse among various AARs. Our results provide a basis for selecting an AAR sequence suitable for metabolic engineering of bioalkane production while regulating carbon chain length.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0644-5) contains supplementary material, which is available to authorized users.

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.Rhodococcus is a widely occurring genus of aerobic, nonmotile soil bacteria that are closely related to three other genera of GC-rich actinomycetes: Gordonia, Nocardia, and Mycobacterium. Rhodococci degrade an extraordinarily wide variety of organic substrates and thus play an important role in the global C cycle. The unusual armamentarium of enzymatic activities involved in these processes has been exploited in applications ranging from commodity chemical production to the desulfurization of fossil fuels (6). Consequently, the metabolic capabilities of rhodococci are of interest to the pharmaceutical, environmental, chemical, and energy sectors.Rhodococcus sp. strain RHA1 is characterized by its exceptional ability to transform polychlorinated biphenyls (PCBs) (53), a particularly widespread and persistent class of environmental pollutants. It is generally thought that in aerobic bacteria, PCBs are cometabolized by the bph pathway, which is responsible for the aerobic degradation of biphenyl (23). The upper bph pathway consists of four enzymatic activities that together transform biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate. For each of these four steps, RHA1 appears to possess multiple isozymes, which may help explain the strain's superior PCB-transforming capabilities. Thus, the strain contains at least three bph-type ring-hydroxylating di...

Background Cyanobacteria produce hydrocarbons corresponding to diesel fuels by means of aldehyde-deformylating oxygenase (ADO). ADO catalyzes a difficult and unusual reaction in the conversion of aldehydes to hydrocarbons and has been widely used for biofuel production in metabolic engineering; however, its activity is low. A comparison of the amino acid sequences of highly active and less active ADOs will elucidate non-conserved residues that are essential for improving the hydrocarbon-producing activity of ADOs. Results Here, we measured the activities of ADOs from 10 representative cyanobacterial strains by expressing each of them in Escherichia coli and quantifying the hydrocarbon yield and amount of soluble ADO. We demonstrated that the activity was highest for the ADO from Synechococcus elongatus PCC 7942 (7942ADO). In contrast, the ADO from Gloeobacter violaceus PCC 7421 (7421ADO) had low activity but yielded high amounts of soluble protein, resulting in a high production level of hydrocarbons. By introducing 37 single amino acid substitutions at the non-conserved residues of the less active ADO (7421ADO) to make its sequence more similar to that of the highly active ADO (7942ADO), we found 20 mutations that improved the activity of 7421ADO. In addition, 13 other mutations increased the amount of soluble ADO while maintaining more than 80% of wild-type activity. Correlation analysis showed a solubility-activity trade-off in ADO, in which activity was negatively correlated with solubility. Conclusions We succeeded in identifying non-conserved residues that are essential for improving ADO activity. Our results may be useful for generating combinatorial mutants of ADO that have both higher activity and higher amounts of the soluble protein in vivo, thereby producing higher yields of biohydrocarbons. Electronic supplementary material The online version of this article (10.1186/s13068-019-1409-8) contains supplementary material, which is available to authorized users.

Cyanobacterial biosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based fuels. Key enzymes for bioalkane production in cyanobacteria are acyl-ACP reductase (AAR) and aldehyde-deformylating oxygenase (ADO). AAR catalyzes the reduction of the fatty acyl-ACP/CoA substrates to fatty aldehydes, which are then converted into alkanes/alkenes by ADO. These enzymes have been widely used for biofuel production by metabolic engineering of cyanobacteria and other organisms. However, both proteins, particularly ADO, have low enzymatic activities, and their catalytic activities are desired to be improved for use in biofuel production. Recently, progress has been made in the basic sciences and in the application of AAR and ADO in alkane production. This chapter provides an overview of recent advances in the study of the structure and function of AAR and ADO, protein engineering of these enzymes for improving activity and modifying substrate specificities, and examples of metabolic engineering of cyanobacteria and other organisms using AAR and ADO for biofuel production.