Polyhydroxyalkanoates (PHAs) are a diverse family of sustainable bioplastics synthesized by various bacteria, but their high production cost and unstable material properties make them challenging to ...use in commercial applications. Current industrial biotechnology (CIB) employs conventional microbial chassis, leading to high production costs. However, next-generation industrial biotechnology (NGIB) approaches, based on fast-growing and contamination-resistant extremophilic Halomonas spp., allow stable continuous processing and thus economical production of PHAs with stable properties. Halomonas spp. designed and constructed using synthetic biology not only produce low-cost intracellular PHAs but also secrete extracellular soluble products for improved process economics. Next-generation industrial biotechnology is expected to reduce the bioproduction cost and process complexity, leading to successful commercial production of PHAs.
The high production cost, poor thermal and mechanical properties, and unstable quality of polyhydroxyalkanoates (PHAs) are the grand challenges to be addressed before industrialization.Next-generation industrial biotechnology (NGIB) based on extremophiles is emerging to meet most of these challenges.Fast-growing and contamination-resistant Halomonas spp. allow open, unsterile, and continuous fermentations to produce PHAs with low-cost and stable properties.Halomonas spp. constructed by synthetic biology generate large sizes for PHA accumulation and gravity separation or coproduction of extracellular soluble products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Next generation industrial biotechnology (NGIB) based on extremophilic bacteria grown under unsterile and continuous way in plastic transparent bioreactors.▪
•Low petroleum prices require innovations ...to make bio-production competitive.•Contamination resistant extremophilic microorganisms can simplify bioprocessing.•Next generation industrial biotechnology (NGIB) is energy and fresh water saving.•NGIB should be operated under open and continuous conditions and easily automated.•NGIB should make bulk chemical production as competitive as chemical processes.
Industrial biotechnology aims to produce bulk chemicals including polymeric materials and biofuels based on bioprocessing sustainable agriculture products such as starch, fatty acids and/or cellulose. However, traditional bioprocesses require bioreactors made of stainless steel, complicated sterilization, difficult and expensive separation procedures as well as well-trained engineers that are able to conduct bioprocessing under sterile conditions, reducing the competitiveness of the bio-products. Amid the continuous low petroleum price, next generation industrial biotechnology (NGIB) allows bioprocessing to be conducted under unsterile (open) conditions using ceramic, cement or plastic bioreactors in a continuous way, it should be an energy, water and substrate saving technology with convenient operation procedure. NGIB also requires less capital investment and reduces demand on highly trained engineers. The foundation for the simplified NGIB is microorganisms that resist contaminations by other microbes, one of the examples is rapid growing halophilic bacteria inoculated under high salt concentration and alkali pH. They have been engineered to produce multiple products in various scales.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
To avoid large open surgery using scaffold transplants, small‐sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and ...viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300–360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10–60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast‐regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier‐free hMSCs in an ectopic bone‐formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro‐Noah's Ark to safely transport cells to a defect tissue.
Combining the advantages of microspheres and scaffolds, highly open porous microspheres (OPMs) made of polyhydroxyalkanoate (PHA) are developed as injectable carriers harboring growing stem cells. The PHA OPMs protect the stem cells from stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues, functioning like a micro‐Noah's Ark to safely transport cells to a designated tissue location for regeneration.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biology and DNA-editing approaches have been adopted to engineer cells ...for more efficient PHA production. Recent advances in synthetic biology applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphology engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biology methods and genome-editing tools contribute to controllable PHA molecular weights and compositions, enhanced PHA accumulation, and easy downstream processing.
The bioplastic PHA, which features biodegradability, biocompatibility, and thermoprocessibility, is moving toward low-cost microbial production to replace nondegradable petrochemical plastics.Wild-type or weakly engineered bacteria are insufficient to meet demands for improved PHA structures and low production cost.Synthetic biology and genome-editing approaches can promote PHA synthesis, enlarge cells for more PHA storage, control shape changes, accelerate growth, aid the co-production of multiple products, direct flux toward final products, and make product recovery more convenient.Optimized promoters and RBSs increase the expression of PHA synthesis genes.CRISPR interference and CRISPR/Cas9 are useful for downregulating the expression of multiple genes simultaneously, allowing more flux to be directed to PHA synthesis in an optimized strain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy ...consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production.
•Industrial biotechnology is less competitive compared with chemical processes.•Halophiles allow fermentation processes to run contamination free under unsterile conditions and continuous way.•Halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants.•Genetic manipulation methods have been developed for halophiles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
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.
Manipulation bacterial cells for maximizing PHA synthesis.▪
•Improvements on PHA biosynthesis must be considered in a systematic way.•Directing metabolic flux to PHA synthesis is critical for ...improving conversion rate.•Engineering rapid growth and cell sizes are important for PHA biosynthesis efficiency.•The newly emerging tools and reprogramming DNA are helping to control PHA biosynthesis.•A suitable chassis for engineering all the above properties is desirable.
Biosynthesis of polyhydroxyalkanoates (PHA) has been studied since the 1920s. The biosynthesis pathways have been well understood and various attempts have been made to improve the PHA biosynthesis efficiency. Recent progresses have been focused on systematic improvements on PHA biosynthesis including changing growth pattern for rapid proliferation, engineering to enlarge cell sizes for more PHA accumulation space, reprogramming the PHA synthesis pathways using optimized RBS and promoter, redirecting metabolic flux to PHA synthesis using CRISPR/Cas9 tools, and very importantly, the employment of non-traditional host such as halophiles for reduced complexity on PHA production. All of the efforts should lead to ultrahigh PHA accumulation, controllable PHA compositions and molecular weights, open and continuous PHA production with gravity separation processes, resulting in competitive PHA production cost.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Clustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has ...been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels.
For the first time CRISPRi was successfully used in Halomonas sp. TD01 to repress expression of ftsZ gene encoding bacterial fission ring formation protein, leading to an elongated cell morphology with typical filamentous shape similar to phenomenon observed with Escherichia coli. CRISPRi was employed to regulate expressions of prpC gene encoding 2-methylcitrate synthase for regulating 3-hydroxyvalerate monomer ratio in PHBV copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Percentages of HV in PHBV copolymers were controllable ranging from less than 1 to 13%. Furthermore, repressions on gltA gene encoding citrate synthase channeled more acetyl-CoA from the tricarboxylic acid (TCA) cycle to poly(3-hydroxybutyrate) (PHB) synthesis. The PHB accumulation by Halomonas sp. TD01 with its gltA gene repressed in various intensities via CRISPRi was increased by approximately 8% compared with the wild type control containing the CRISPRi vector without target.
It has now been confirmed that the CRISPRi system can be applied to Halomonas sp. TD01, a promising industrial strain for production of various PHA and chemicals under open and continuous fermentation process conditions. In details, the CRISPRi system was successfully designed in this study to target genes of ftsZ, prpC and gltA, achieving longer cell sizes, channeling more substrates to PHBV and PHB synthesis, respectively. CRISPRi can be expected to use for more metabolic engineering applications in non-model organisms.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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 (AldD
), 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
of 3HP with a yield and productivity of 0.93 g g
1,3-propanediol and 2.4 g L
h
, 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.
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