Synthetic Biology Approaches to Hydrocarbon Biosensors: A Review

Monooxygenases are a class of enzymes that facilitate the bacterial degradation of alkanes and alkenes. The regulatory components associated with monooxygenases are nature’s own hydrocarbon sensors, and once functionally characterised, these components can be used to create rapid, inexpensive and sensitive biosensors for use in applications such as bioremediation and metabolic engineering. Many bacterial monooxygenases have been identified, yet the regulation of only a few of these have been investigated in detail. A wealth of genetic and functional diversity of regulatory enzymes and promoter elements still remains unexplored and unexploited, both in published genome sequences and in yet-to-be-cultured bacteria. In this review we examine in detail the current state of research on monooxygenase gene regulation, and on the development of transcription-factor-based microbial biosensors for detection of alkanes and alkenes. A new framework for the systematic characterisation of the underlying genetic components and for further development of biosensors is presented, and we identify focus areas that should be targeted to enable progression of more biosensor candidates to commercialisation and deployment in industry and in the environment.

Long-Term Adaptation of Acidophilic Archaeal Ammonia Oxidisers Following Different Soil Fertilisation Histories

Ammonia oxidising archaea (AOA) are ecologically important nitrifiers in acidic agricultural soils. Two AOA phylogenetic clades, belonging to order-level lineages of Nitrososphaerales (clade C11; also classified as NS-Gamma-2.3.2) and family-level lineage of Candidatus Nitrosotaleaceae (clade C14; NT-Alpha-1.1.1), usually dominate AOA population in low pH soils. This study aimed to investigate the effect of different fertilisation histories on community composition and activity of acidophilic AOA in soils. High-throughput sequencing of ammonia monooxygenase gene (amoA) was performed on six low pH agricultural plots originating from the same soil but amended with different types of fertilisers for over 20 years and nitrification rates in those soils were measured. In these fertilised acidic soils, nitrification was likely dominated by Nitrososphaerales AOA and ammonia-oxidising bacteria, while Ca. Nitrosotaleaceae AOA activity was non-significant. Within Nitrososphaerales AOA, community composition differed based on the fertilisation history, with Nitrososphaerales C11 only representing a low proportion of the community. This study revealed that long-term soil fertilisation selects for different acidophilic nitrifier communities, potentially through soil pH change or through direct effect of nitrogen, potassium and phosphorus. Comparative community composition among the differently fertilised soils also highlighted the existence of AOA phylotypes with different levels of stability to environmental changes, contributing to the understanding of high AOA diversity maintenance in terrestrial ecosystems.

Improved 11α-hydroxycanrenone production by modification of cytochrome P450 monooxygenase gene in Aspergillus ochraceus

Eplerenone is a drug that protects the cardiovascular system. 11α-Hydroxycanrenone is a key intermediate in eplerenone synthesis. We found that although the cytochrome P450 (CYP) enzyme system in Aspergillus ochraceus strain MF018 could catalyse the conversion of canrenone to 11α-hydroxycanrenone, its biocatalytic efficiency is low. To improve the efficiency of 11α-hydroxycanrenone production, the CYP monooxygenase-coding gene of MF018 was predicted and cloned based on whole-genome sequencing results. A recombinant A. ochraceus strain MF010 with the high expression of CYP monooxygenase was then obtained through homologous recombination. The biocatalytic rate of this recombinant strain reached 93 % at 60 h without the addition of organic solvents or surfactants and was 17–18 % higher than that of the MF018 strain. Moreover, the biocatalytic time of the MF010 strain was reduced by more than 30 h compared with that of the MF018 strain. These results show that the recombinant A. ochraceus strain MF010 can overcome the limitation of substrate biocatalytic efficiency and thus holds a high poten tial for application in the industrial production of eplerenone.

Functional Analysis of P450 Monooxygenase SrrO in the Biosynthesis of Butenolide-Type Signaling Molecules in Streptomyces rochei

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics lankacidin and lankamycin, and their biosynthesis is tightly controlled by butenolide-type signaling molecules SRB1 and SRB2. SRBs are synthesized by SRB synthase SrrX, and induce lankacidin and lankamycin production at 40 nM concentration. We here investigated the role of a P450 monooxygenase gene srrO (orf84), which is located adjacent to srrX (orf85), in SRB biosynthesis. An srrO mutant KA54 accumulated lankacidin and lankamycin at a normal level when compared with the parent strain. To elucidate the chemical structures of the signaling molecules accumulated in KA54 (termed as KA54-SRBs), this mutant was cultured (30 L) and the active components were purified. Two active components (KA54-SRB1 and KA54-SRB2) were detected in ESI-MS and chiral HPLC analysis. The molecular formulae for KA54-SRB1 and KA54-SRB2 are C15H26O4 and C16H28O4, whose values are one oxygen smaller and two hydrogen larger when compared with those for SRB1 and SRB2, respectively. Based on extensive NMR analysis, the signaling molecules in KA54 were determined to be 6′-deoxo-SRB1 and 6′-deoxo-SRB2. Gel shift analysis indicated that a ligand affinity of 6′-deoxo-SRB1 to the specific receptor SrrA was 100-fold less than that of SRB1. We performed bioconversion of the synthetic 6′-deoxo-SRB1 in the Streptomyces lividans recombinant carrying SrrO-expression plasmid. Substrate 6′-deoxo-SRB1 was converted through 6′-deoxo-6′-hydroxy-SRB1 to SRB1 in a time-dependent manner. Thus, these results clearly indicated that SrrO catalyzes the C-6′ oxidation at a final step in SRB biosynthesis.

Complete Genome of Isoprene Degrading Nocardioides sp. WS12

Isoprene is a climate-active gas whose wide-spread global production stems mostly from terrestrial plant emissions. The biodegradation of isoprene is carried out by a number of different bacteria from a wide range of environments. This study investigates the genome of a novel isoprene degrading bacterium Nocardioides sp. WS12, isolated from soil associated with Salix alba (Willow), a tree known to produce high amounts of isoprene. The Nocardioides sp. WS12 genome was fully sequenced, revealing the presence of a complete isoprene monooxygenase gene cluster, along with associated isoprene degradation pathway genes. Genes associated with rubber degradation were also present, suggesting that Nocardioides sp. WS12 may also have the capacity to degrade poly-cis-1,4-isoprene.

Degradation Potential of the Nonylphenol Monooxygenase of Sphingomonas sp. NP5 for Bisphenols and Their Structural Analogs

The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene (nmoA) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4′-dihydroxybenzophenone, 4,4′-dihydroxydiphenylether, and 4,4′-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure.