Utilization of gene manipulation system for advancing the biotechnological potential of halophiles: A review.

Halophiles are salt-loving microorganisms known to have their natural resistance against media contamination even when cultivated in nonsterile and continuous bioprocess system, thus acting as promising cell factories for Next Generation of Industrial Biotechnology (NGIB). NGIB - a successor to the traditional industrial biotechnology, is a more sustainable and efficient bioprocess technology while saving energy and water in a more convenient way as well as reducing the investment cost and skilled workforce requirement. Numerous studies have achieved intriguing outcomes during synthesis of different metabolite using halophiles such as polyhydroxyalkanoates (PHA), ectoine, biosurfactants, and carotenoids. Present-day development in genetic maneuverings have shown optimistic effects on the industrial applications of halophiles. However, viable and competent genetic manipulation system and gene editing tools are critical to accelerate the process of halophile engineering. With the aid of such powerful gene manipulation systems, exclusive microbial chassis are being crafted with desirable features to breed another innovative area of research such as synthetic biology. This review provides an aerial perspective on how the expansion of adaptable gene manipulation toolkits in halophiles are contributing towards biotechnological advancement, and also focusses on their subsequent application for production improvement. This current methodical and comprehensive review will definitely help the scientific fraternity to bridge the gap between challenges and opportunities in halophile engineering.

Cupriavidus necator as a platform for polyhydroxyalkanoate production: An overview of strains, metabolism, and modeling approaches.

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.

Food wastes can be hydrolyzed into soluble microbial substrates, contributing to sustainability. Halomonas spp.-based Next Generation Industrial Biotechnology (NGIB) allows open, unsterile fermentation, eliminating the need for sterilization to avoid the Maillard reaction that negatively affects cell growth. This is especially important for food waste hydrolysates, which have a high nutrient content but are unstable due to batch, sources, or storage conditions. These make them unsuitable for polyhydroxyalkanoate (PHA) production, which usually requires limitation on either nitrogen, phosphorous, or sulfur. In this study, H. bluephagenesis was constructed by overexpressing the PHA synthesis operon phaCABCn (cloned from Cupriavidus necator) controlled by the essential gene ompW (encoding outer membrane protein W) promoter and the constitutive porin promoter that are continuously expressed at high levels throughout the cell growth process, allowing poly(3-hydroxybutyrate) (PHB) production to proceed in nutrient-rich (also nitrogen-rich) food waste hydrolysates of various sources. The recombinant H. bluephagenesis termed WZY278 generated 22 g L-1 cell dry weight (CDW) containing 80 wt% PHB when cultured in food waste hydrolysates in shake flasks, and it was grown to 70 g L-1 CDW containing 80 wt% PHB in a 7-L bioreactor via fed-batch cultivation. Thus, unsterilizable food waste hydrolysates can become nutrient-rich substrates for PHB production by H. bluephagenesis able to be grown contamination-free under open conditions.

Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas.

Ligand-induced system plays an important role for microbial engineering due to its tunable gene expression control over timings and levels. An oleic acid (OA)-induced system was recently constructed based on protein FadR, a transcriptional regulator involved in fatty acids metabolism, for metabolic control in Escherichia coli. In this study, we constructed a synthetic FadR-based OA-induced systems in Halomonas bluephagenesis by hybridizing the porin promoter core region and FadR-binding operator (fadO). The dynamic control range was optimized over 150-fold, and expression leakage was significantly reduced by tuning FadR expression and positioning fadO, forming a series of OA-induced systems with various expression strengths, respectively. Additionally, ligand orthogonality and cross-species portability were also studied and showed highly linear correlation among Halomonas spp., Escherichia coli and Pseudomonas spp. Finally, OA-induced systems with medium- and small-dynamic control ranges were employed to dynamically control the expression levels of morphology associated gene minCD, and monomer precursor 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathway for polyhydroxyalkanoates (PHA), respectively, in the presence of oleic acid as an inducer. As a result, over 10 g/L of poly-3-hydroxybutyrate (PHB) accumulated by elongated cell sizes, and 6 g/L of P(3HB-co-9.57 mol% 4HB) were obtained by controlling the dose and induction time of oleic acid only. This study provides a systematic approach for ligand-induced system engineering, and demonstrates an alternative genetic tool for dynamic control of industrial biotechnology.

Biosynthesis of diverse α,ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis.

Polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible plastics with potential to replace petroleum based plastics. Diversity of PHA monomer structures provides flexibility in material properties to suit more applications. In this study, 5-hydroxyvalerate (5HV) synthesis pathway was established based on intrinsic alcohol/aldehyde dehydrogenases. The PHA polymerase cloned from Cupriavidus necator functions to polymerize 5HV into its copolymers in ratios ranging from 8% to 32%. Elastic copolymer P(85% 3HB-co-15% 5HV) was generated with an elongation at break and a Young's modulus of 1283% and 73.1 MPa, respectively. The recombinant H. bluephagenesis was able to convert various diols including 1, 3-propanediol, 1, 4-butanediol and 1, 5-pentanediol into PHA, leading to 13 PHA polymers including transparent P(53% 3HB-co-20% 4HB-co-27% 5HV) and sticky P(3HB-co-3HP-co-4HB-co-5HV). The engineered H. bluephagenesis was successfully grown in a 7-L bioreactor to produce the highly elastic P(85% 3HB-co-15% 5HV) and the sticky P(3HB-co-3HP-co-4HB-co-5HV), demonstrating their potential for industrial scale-up.

Metabolic Engineering of Escherichia coli for Production of Polyhydroxyalkanoates with Hydroxyvaleric Acid Derived from Levulinic Acid.

Polyhydroxyalkanoates (PHAs) are emerging as alternatives to plastics by replacing fossil fuels with renewable raw substrates. Herein, we present the construction of engineered Escherichia coli strains to produce short-chain-length PHAs (scl-PHAs), including the monomers 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) produced from levulinic acid (LA). First, an E. coli strain expressing genes (lvaEDABC) from the LA metabolic pathway of Pseudomonas putida KT2440 was constructed to generate 4HV-CoA and 3HV-CoA. Second, both PhaAB enzymes from Cupriavidus necator H16 were expressed to supply 3-hydroxybutyrate (3HB)-CoA from acetyl-CoA. Finally, PHA synthase (PhaCCv) from Chromobacterium violaceum was introduced for the subsequent polymerization of these three monomers. The resulting E. coli strains produced four PHAs (w/w% of dry cell weight): 9.1 wt% P(4HV), 1.7 wt% P(3HV-co-4HV), 24.2 wt% P(3HB-co-4HV), and 35.6 wt% P(3HB-co-3HV-co-4HV).

Complete Genome Sequence of the Type Strain Tepidimonas taiwanensis LMG 22826T, a Thermophilic Alkaline Protease and Polyhydroxyalkanoate Producer.

Tepidimonas taiwanensis is a moderately thermophilic, Gram-negative, rod-shaped, chemoorganoheterotrophic, motile bacterium. The alkaline protease producing type strain T. taiwanensis LMG 22826T was recently reported to also be a promising producer of polyhydroxyalkanoates (PHAs)-renewable and biodegradable polymers representing an alternative to conventional plastics. Here, we present its first complete genome sequence which is also the first complete genome sequence of the whole species. The genome consists of a single 2,915,587-bp-long circular chromosome with GC content of 68.75%. Genome annotation identified 2,764 genes in total while 2,634 open reading frames belonged to protein-coding genes. Although functional annotation of the genome and division of genes into Clusters of Orthologous Groups (COGs) revealed a relatively high number of 694 genes with unknown function or unknown COG, the majority of genes were assigned a function. Most of the genes, 406 in total, were involved in energy production and conversion, and amino acid transport and metabolism. Moreover, particular key genes involved in the metabolism of PHA were identified. Knowledge of the genome in connection with the recently reported ability to produce bioplastics from the waste stream of wine production makes T. taiwanensis LMG 22826T, an ideal candidate for further genome engineering as a bacterium with high biotechnological potential.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Complete genome sequence of Photobacterium ganghwense C2.2: A new polyhydroxyalkanoate production candidate.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based plastics. Here, we describe the complete genome of a new marine PHA-producing bacterium-Photobacterium ganghwense (strain C2.2), which we have isolated from the Black Sea seashore. This new isolate is psychrotolerant and accumulates PHA when glycerol is provided as the main carbon source. Transmission electron microscopy, specific staining with Nile Red visualized via epifluorescence microscopy and gas chromatography analysis confirmed the accumulation of PHA. This is the only PHA-producing Photobacterium for which we now have a complete genome sequence, allowing us to investigate the pathways for PHA production and other secondary metabolite synthesis pathways. The de novo assembly genome, obtained using open-source tools, comprises two chromosomes (3.5, 2 Mbp) and a megaplasmid (202 kbp). We identify the entire PHA synthesis gene cluster that encodes a class I PHA synthase, a phasin, a 3-ketothiolase, and an acetoacetyl-CoA reductase. No conventional PHA depolymerase was identified in strain C2.2, but a putative lipase with extracellular amorphous PHA depolymerase activity was annotated, suggesting that C2.2 is unable to degrade intracellular PHA. A complete pathway for the conversion of glycerol to acetyl-CoA was annotated, in accordance with its ability to convert glycerol to PHA. Several secondary metabolite biosynthetic gene clusters and a low number of genes involved in antibiotic resistance and virulence were also identified, indicating the strain's suitability for biotechnological applications.

Engineering of Shewanella marisflavi BBL25 for biomass-based polyhydroxybutyrate production and evaluation of its performance in electricity production.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Metabolic engineering of Methylorubrum extorquens AM1 for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production using formate.

Formate is a promising environmentally friendly and sustainable feedstock synthesized from syngas or carbon dioxide. Methylorubrum extorquens is a type II methylotroph that can use formate as a carbon source. It accumulates polyhydroxyalkanoates (PHAs) inside the cell, mainly producing poly-3-hydroxybutyrate (PHB), a degradable biopolymer. Owing to its high melting point and stiff nature, however, mechanical property improvement is warranted in the form of copolymerization. To produce the PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the endogenous gene phaC was deleted and the pathway genes bktB, phaJ1, and phaC2, with broader substrate specificities, were heterologously expressed. To improve the incorporation of 3-hydroxyvalerate (3HV), the expression level of bktB was improved by untranslated region (UTR) engineering, and the endogenous gene phaA was deleted. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, when propionate and butyrate were supplemented, PHBVs with 3HV portions of up to 70.6% were produced. This study shows that a PHBV copolymer with a high proportion of 3HV can be synthesized using formate, a C1 carbon source, through metabolic engineering and supplementation with short-chain fatty acids.

Modifying the Cyanobacterial Metabolism as a Key to Efficient Biopolymer Production in Photosynthetic Microorganisms.

Cyanobacteria are photoautotrophic bacteria commonly found in the natural environment. Due to the ecological benefits associated with the assimilation of carbon dioxide from the atmosphere and utilization of light energy, they are attractive hosts in a growing number of biotechnological processes. Biopolymer production is arguably one of the most critical areas where the transition from fossil-derived chemistry to renewable chemistry is needed. Cyanobacteria can produce several polymeric compounds with high applicability such as glycogen, polyhydroxyalkanoates, or extracellular polymeric substances. These important biopolymers are synthesized using precursors derived from central carbon metabolism, including the tricarboxylic acid cycle. Due to their unique metabolic properties, i.e., light harvesting and carbon fixation, the molecular and genetic aspects of polymer biosynthesis and their relationship with central carbon metabolism are somehow different from those found in heterotrophic microorganisms. A greater understanding of the processes involved in cyanobacterial metabolism is still required to produce these molecules more efficiently. This review presents the current state of the art in the engineering of cyanobacterial metabolism for the efficient production of these biopolymers.

Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina.

Pseudomonas mendocina NK-01 previously isolated by our lab is able to accumulate medium-chain-length polyhydroxyalkanoate (mcl-PHA) intracellularly and secrete alginate oligosaccharide (AO) to the extracellular milieu. The present study aimed at investigating whether improved production of mcl-PHA and AO by P. mendocina can be accomplished by genome reduction. In this study, 14 large genomic fragments accounting for 7.7% of the genome of P. mendocina NK-01 were sequentially deleted to generate a series of genome-reduced strains by an upp-based markerless knockout method. As a result, the intracellular ATP/ADP ratio of the strain NKU421 with the largest deletion improved by 11 times compared to NK-01. More importantly, the mcl-PHA and AO yields of NKU421 increased by 114.8% and 27.8%, respectively. Enhancing mcl-PHA and AO production by NKU421 may be attributed to improved transcriptional levels of PHA synthase genes and AO secretion-related genes. The present study suggests that rational reduction of bacterial genome is a feasible approach to construct an optimal chassis for enhanced production of bacterial metabolites. In the future, further reduction of the NKU421 genome can be expected to create high-performance chassis for the development of microbial cell factories.

Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and phaJ genes in Cupriavidus necator.

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.

Biodiversity and Habitats of Polar Region Polyhydroxyalkanoic Acid-Producing Bacteria: Bioprospection by Popular Screening Methods.

Polyhydroxyalkanoates (PHAs), the intracellular polymers produced by various microorganisms as carbon and energy storage, are of great technological potential as biodegradable versions of common plastics. PHA-producing microbes are therefore in great demand and a plethora of different environments, especially extreme habitats, have been probed for the presence of PHA-accumulators. However, the polar region has been neglected in this regard, probably due to the low accessibility of the sampling material and unusual cultivation regime. Here, we present the results of a screening procedure involving 200 bacterial strains isolated from 25 habitats of both polar regions. Agar-based tests, microscopy, and genetic methods were conducted to elucidate the biodiversity and potential of polar-region PHA-accumulators. Microscopic observation of Nile Red stained cells proved to be the most reliable screening method as it allowed to confirm the characteristic bright orange glow of the Nile Red-PHA complex as well as the typical morphology of the PHA inclusions. Psychrophilic PHA-producers belonged mostly to the Comamonadaceae family (Betaproteobacteria) although actinobacterial PHA synthesizers of the families, Microbacteriaceae and Micrococcaceae also featured prominently. Glacial and postglacial habitats as well as developed polar region soils, were evaluated as promising for PHA-producer bioprospection. This study highlights the importance of psychrophiles as biodiverse and potent polyhydroxyalkanoate sources for scientific and application-aimed research.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions.

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Using biopolymer bodies for encapsulation of hydrophobic products in bacterium.

Producing some small hydrophobic molecules in microbes is challenging. Often these molecules cannot cross membranes, and thus their production may be limited by lack of storage space in the producing organism. This study reports a new technology for in vivo storage of valuable hydrophobic products in/on biopolymer bodies in Escherichia coli. A biodegradable and biocompatible polyester - poly (3-hydroxybutyrate) (PHB) - was selected as the intracellular storage vessel to encapsulate lycopene, which is a chromogenic model compound. The hydrophobic interaction between lycopene and PHB was verified by using in vitro binding test and sucrose density gradient centrifugation. Further in vivo characterization was performed by using Confocal Laser Scanning Microscopy (CLSM). The images validated the in vivo co-localization between PHB granules and lycopene. The images also showed that lycopene aggregated in bacteria that did not produce PHB, which may challenge the commonly accepted hypothesis that most lycopene molecules are stored in cell membranes of recombinant host. We also confirmed that producing PHB did not negatively affect lycopene biosynthesis in the E. coli strains and collected data suggesting that PHB titer and lycopene titer were positively correlated when the cells were engineered to co-produce them. The biopolymers that encapsulated hydrophobic molecules could have many useful applications, especially in controlled release because the polymers are biodegradable, and the encapsulated products would be released during the polymer degradation.

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis.

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.

Enhanced rhamnolipids production in Pseudomonas aeruginosa SG by selectively blocking metabolic bypasses of glycosyl and fatty acid precursors.

OBJECTIVE: To enhance rhamnolipids production in Pseudomonas aeruginosa, an optimization strategy based on selectively blocking the metabolic bypass that competed precursors with rhamnolipids biosynthesis pathway, containing exopolysaccharide (Psl and Pel) and polyhydroxyalkanoates (PHA) synthesis pathways. RESULTS: Blocking the synthesis of Psl and PHA by genes knockout, both mutants P. aeruginosa SG ∆pslAB and P. aeruginosa SG ∆phaC1DC2 can grow normally in fermentation medium and increase the production of rhamnolipids by 21% and 25.3%, respectively. While blocking the synthesis of Pel, the cell growth of the mutant strain P. aeruginosa SG ∆pelA was inhibited, thus its production yield of rhamnolipids was also decreased by 39.8%. In addition, simultaneously blocking the synthesis of Psl and PHA, a double mutant strain P. aeruginosa SG ∆pslAB ∆phaC1DC2 was constructed. Rhamnolipids production was significantly increased in strain SG ∆pslAB ∆phaC1DC2 by 69.7%. CONCLUSION: Through selectively blocking metabolic bypasses, increasing the amount of glycosyl and fatty acid precursors can significantly enhance rhamnolipids production in P. aeruginosa.