Project description:Biomanufacturing remains financially uncompetitive with the lower cost but higher carbon emitting hydrocarbon based chemical industry. Novel chassis organisms may enable cost reductions with respect to traditional chassis such as E. coli and so open an economic rout to low emission biomanufacturing. Extremophile bacteria exemplify that potential. Salt tolerant halomonas species thrive in conditions inimical to other organisms. Their adoption would eliminate the cost of sterilising equipment. Novel chassis are inevitably poorly understood in comparison to established organisms. Rapid characterisation and community data sharing will facilitate organisms’ adoption for biomanufacturing. This paper describes baseline proteomics data set for Halomonas bluephagenesis TD01 under active development for biomanufactoring. The data record comprises a newly sequenced genome for the organism; evidence for expression of 1150 proteins (30% of the proteome) including baseline quantification of 1050 proteins (27% of the proteome) and a spectral library enabling re-use for targeted proteomics assays. Protein data is annotated with KEGG Orthology enabling rapid matching of quantitative data to pathways of interest to biomanufacturing.
Project description:3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L-1 of 3HP with a yield and productivity of 0.93 g g-1 1,3-propanediol and 2.4 g L-1 h-1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32-45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.
Project description:Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L-1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L-1 ectoine and 32 g L-1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.
Project description:Halomonas bluephagenesis, a haloalkaliphilic bacterium and native polyhydroxybutyrate (PHB) producer, is a non-traditional bioproduction chassis for the next generation industrial biotechnology (NGIB). A single-sgRNA CRISPR/Cas9 genome editing tool is optimized using dual-sgRNA strategy to delete large DNA genomic fragments (>50 kb) with efficiency of 12.5% for H. bluephagenesis. The non-essential or redundant gene clusters of H. bluephagenesis, including those encoding flagella, exopolysaccharides (EPSs) and O-antigen, are sequentially deleted using this improved genome editing strategy. Totally, ~3% of the genome is reduced with its rapid growth and high PHB-production ability unaffected. The deletion of EPSs and O-antigen gene clusters shows two excellent properties from industrial perspective. Firstly, the EPSs and O-antigen deleted mutant rapidly self-flocculates and precipitates within 20 min without centrifugation. Secondly, DNA transformation into the mutant using electroporation becomes feasible compared to the wild-type H. bluephagenesis. The genome-reduced H. bluephagenesis mutant reduces energy and carbon source requirement to synthesize PHB comparable to its wild type. The H. bluephagenesis chassis with a reduced genome serves as an improved version of a NGIB chassis for productions of polyhydroxyalkanoates (PHA) or other chemicals.