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BackgroundPropane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli.ResultsWe report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L).ConclusionsThis study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde.Graphical abstractPropane synthesis derived from a fermentative butanol pathway is enabled by metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0231-1) contains supplementary material, which is available to authorized users.

Nature's repertoire of bio-halogenase enzymes is intriguing with halogenases from various natural product biosynthetic clusters that carry out site and region-specific halogenation of diverse bioactive precursors and molecules. Currently, we have a comprehensive catalogue of cryptic and non-cryptic halogenases that act on simple to complex aliphatic and aromatic molecular scaffolds. This will open up further synthetic and biosynthetic opportunities for C-H activation, ring formation and functionalization of different molecular structures. In fact, halogenases were exploited over the years for these potential applications, to replace traditional chemical halogenation chemistries toward creating economical and environmentally benign methodologies and also for biosynthetic pathways. This review will discuss our advances in utilizing biohalogenases to generate both in vivo and in vitro biosynthetic pathways; summarizing all naturals and non-naturals that are synthesized with a direct bio-halogenase incorporation.

The development of enzymatic routes toward amide and carboxylic acid bond formation in bioactive molecular scaffolds using aqueous conditions is a major challenge for biopharmaceutical and fine chemical industrial sectors. We report biocatalytic and kinetic characterization of two indole-3-acetamide (IAM) pathway enzymes, tryptophan-2-monooxygenase (iaaM) and indole-3-acetamide hydrolase (iaaH), present in plant microbiomes that produce indole-3-acetic acid (IAA). In this pathway, tryptophan is converted to indole-3-acetamide by the monooxygenase activity of iaaM, followed by its hydrolysis to form carboxylic acid by iaaH enzyme. Since IAA or auxin is an essential natural plant hormone and an important synthon for fine chemicals, the developed monooxygenase-based bioconversion route has a wider scope compared to currently available synthetic and biocatalytic methods to produce synthetic auxins and a range of amides and carboxylic acids for agrochemical and pharmaceutical applications. To display this, one-pot multienzyme biosynthetic cascades for preparativescale production of IAA derivatives were performed by incorporating tryptophan synthase and tryptophan halogenase enzymes. We also report the creation of an efficient de novo biosynthesis for IAA and its derivatives from glucose or indoles via a reconstructed IAM pathway in Escherichia coli.