From Springer Nature via Jisc Publications RouterHistory: received 2020-05-21, accepted 2020-07-08, registration 2020-07-08, pub-electronic 2020-07-14, online 2020-07-14, collection 2020-12Publication status: PublishedFunder: C3 Biotechnologies LtdFunder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/S01778X/1, EP/J020192/1Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/M017702/1, BB/L010798/1Funder: Newton-Mosharafa fundAbstract: Background: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to clean-burning and non-fossil fuel-derived gaseous alkanes (known as ‘bio-LPG’). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of Halomonas (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends). Results: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in E. coli for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into Halomonas strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into Halomonas generated production strains shown to be stable for up to 7 days. Conclusions: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable Halomonas production strains could lead to gas production in the field under non-sterile conditions following process optimisation
