Oxidative Cascade Iodocyclization of 1,n-Dienes: Synthesis of Iodinated Benzo[b]azepine and Benzo[b]azocine Derivatives.

An oxidative cascade iodocyclization of 1,7- or 1,8-dienes has been realized under mild conditions. By employing I2 as an iodine source, this protocol provides a concise and efficient approach to a great deal of biologically significant iodinated benzo[b]azepine and benzo[b]azocine derivatives in moderate to good yields. The gram-scale synthesis and further transformation of products render the approach practical and attractive. Radical trapping and deuterium-labeling experiments help to understand the mechanism.

Photo- or Electrochemical Cyclization of Dienes with Diselenides to Access Seleno-Benzo[b]azepines.

A cascade selenylation/cyclization of dienes with diselenides has been realized under visible-light irradiation or electrolysis conditions. Employing O2 or electricity as a "green" oxidant, this protocol provides a green and efficient method for an array of biologically important seleno-benzo[b]azepine derivatives in moderate to good yields. The direct sunlight irradiation and gram-scale reaction render the approach practical and attractive.

A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts.

Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction.

PHA, a family of natural biopolymers aiming to replace non-degradable plastics for short-term usages, has been developed to include various structures such as short-chain-length (scl) and medium-chain-length (mcl) monomers as well as their copolymers. However, PHA market has been grown slowly since 1980s due to limited variety with good mechanical properties and the high production cost. Here, we review most updated strategies or approaches including metabolic engineering, synthetic biology and morphology engineering on expanding PHA diversity, reducing production cost and enhancing PHA production. The extremophilic Halomonas spp. are taken as examples to show the feasibility and challenges to develop next generation industrial biotechnology (NGIB) for producing PHA more competitively.

Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Manipulation of polyhydroxyalkanoate granular sizes in Halomonas bluephagenesis.

Bacterial polyhydroxyalkanoates (PHA) are a family of intracellular polyester granules with sizes ranging from 100 to 500 nm. Due to their small sizes, it has been very difficult to separate the PHA granules from the bacterial broths. This study aims to engineer the PHA size control mechanism to obtain large PHA granular sizes beneficial for the separation. It has been reported that phasin (PhaP) is an amphiphilic protein located on the surface of PHA granules functioning to regulate sizes and numbers of PHA granules in bacterial cells, deletions on PhaPs result in reduced PHA granule number and enhanced granule sizes. Three genes phaP1, phaP2 and phaP3 encoding three PhaP proteins were deleted in various combinations in halophilic bacterium Halomonas bluephagenesis TD01. The phaP1-knockout strain generated much larger PHA granules with almost the same size as their producing cells without significantly affecting the PHA accumulation yet with a reduced PHA molecular weights. In contrast, the phaP2- and phaP3-knockout strains produced slightly larger sizes of PHA granules with increased PHA molecular weights. While PHA accumulation by phaP3-knockout strains showed a significant reduction. All of the PhaP deletion efforts could not form PHA granules larger than a normal size of H. bluephagenesis TD01. It appears that the PHA granular sizes could be limited by bacterial cell sizes. Therefore, genes minC and minD encoding proteins that block formation of cell fission rings (Z-rings) were over-expressed in various phaP deleted H. bluephagenesis TD01, resulting in large cell sizes of H. bluephagenesis TD01 containing PHA granules with sizes of up to 10 µm that has never been observed previously. It can be concluded that PHA granule sizes are limited by the cell sizes. By engineering a large cell morphology large PHA granules can be produced by PhaP deleted mutants.

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.

Synthesis and Characterization of Polyhydroxyalkanoate Organo/Hydrogels.

Synthetic organogels/hydrogels are attracting growing interests due to their potential applications in biomedical fields, organic electronics, and photovoltaics. Photogelation methods for synthesis of organogels/hydrogels have been shown particularly promising because of the high efficiency and simple synthetic procedures. This study synthesized new biodegradable polyhydroxyalkanoates (PHA)-based organogels/hydrogels via UV photo-cross-linking using unsaturated PHA copolymer poly[(R)-3-hydroxyundecanoate-co-(R)-3-hydroxy-10-undecenoate] (PHU10U) with polyethylene glycol dithiol (PDT) as a photo-cross-linker. The PHU10U was synthesized by an engineered Pseudomonas entomophila and characterized via Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), and 13C NMR. With decreasing the molar ratio of PHU10U to PDT, both the swelling ratio and pore size were decreased. Meanwhile, increasing densities of the gel networks resulted in a higher compressive modulus. Cell cytotoxicity studies based on the CCK-8 assay on both the PHU10U precursor and PHU10U/PDT hydrogels showed that the novel PHA-based biodegradables acting as hydrogels possess good biocompatibility.

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.

Controlling microbial PHB synthesis via CRISPRi.

Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47-75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

Using micro-patterned surfaces to inhibit settlement and biofilm formation by Bacillus subtilis.

Biofilm is a biological complex caused by bacteria attachment to the substrates and their subsequent reproduction and secretion. This phenomenon reduces heat transfer efficiency and causes significant losses in treated sewage heat-recovering systems. This paper describes a physical approach to inhibit bacteria settlement and biofilm formation by Bacillus subtilis, which is the dominant species in treated sewage. Here, micro-patterned surfaces with different characteristics (stripe and cube) and dimensions (1-100 µm) were fabricated as surfaces of interest. Model sewage was prepared and a rotating coupon device was used to form the biofilms. Precision balance, scanning electron microscopy, and confocal laser scanning microscopy (CLSM) were employed to investigate the inhibitory effects and the mechanisms of the biofilm-surface interactions. The results have shown that surfaces with small pattern sizes (1 and 2 µm) all reduced biofilm formation significantly. Interestingly, the CLSM images showed that the surfaces do not play a role in "killing" the bacteria. These findings are useful for future development of new process surfaces on which bacteria settlement and biofilm formation can be inhibited or minimized.

[Effects of Huatan Tongluo Recipe on IL-1ß-induced Proliferation of Rheumatoid Arthritis Synovial Fibroblasts and the Production of TNF-α and aFGF].

Objective To observe the effects of Huatan Tongluo Recipe (HTTLR) on the proliferation of IL-ß p induced rheumatoid arthritis synovial fibroblast ( RASFB) and secretion of necrosis factor α (TNF-α) and acidic fibroblast growth factor (aFGF) in vitro. Methods RASFB cell line was cultured in vitro and stimulated by IL-1ß. The proliferation of RASFB was detected using WST-1 after adding IL-1ß with final concentrations of 1 , 5, 10, 20 µg/L for 24 and 48 h respectively. Then 20 µg/L IL-1ß recruited as induction dose was set up as IL-1ß group. High, middle, low dose HTTLR groups were set up by adding HT- TLR decoction with final concentration of 5%, 2%, 1% (V/V) , respectively for 24 and 48 h. A blank con- trol group was also set up. The proliferation rates were compared. Contents of TNF-α and aFGF were detected in each group using ELISA. mRNA expressions of TNF-α and aFGF were detected using RT-PCR. Results The proliferation rates of RASFB at 24 h and 48 h were lower at 1 µg/L IL-1 ß than at 5, 10, 20 µg/L IL-1ß (P <0. 01). The proliferation rate of RASFB was higher at 10 and 20 µg/L IL-1ß than at 5 µg/L IL-1ß (P <0. 01). Besides, the proliferation rate of RASFB was higher at 20 µg/L IL-1ß than at 10 µg/L IL-1 ß (P <0. 01). The proliferation rate of RASFB was higher at 48 h than at 24 h (P <0. 01). Com- pared with the high dose HTTLR group, the proliferation rate of RASFB was lowered in middle and low dose HTTLR groups (P <0. 01). Besides, IL-1ß induced proliferation rate of RASFB was obviously reduced in the middle dose HTTLR group (P <0. 01). Compared with the blank control group, mRNA ex- pressions of TNF-α and aFGF and their contents were elevated in the IL-1ß group at 24 and 48 h (P < 0. 05). Compared with the IL-1 ß group, mRNA expressions of TNF-α- and aFGF and their contents, except TNF-α- mRNA expression in the low dose HTTLR group at 24 h, were all obviously lowered in 3 dose HTTLR groups at 24 h and 48 h (P <0. 05). Compared with the high dose HTTLR group, mRNA expressions of TNF-(α and aFGF increased in middle and low dose HTTLR groups at 24 h and 48 h; TNF-α content in the low dose HTTLR group at 24 h; contents of TNF-α and aFGF in middle and low dose HTTLR groups at 24 h and 48 h all increased (P <0. 05). Conclusion The mechanism of HTTLR treatment for RA might be related to inhibiting RASFB proliferation, and decreasing mRNA expressions of TNF-α and aFGF as well as their protein secretion.

Enhanced production of polyhydroxybutyrate by multiple dividing E. coli.

BACKGROUND: Most bacteria are grown in a binary fission way meaning a bacterial cell is equally divided into two. Polyhydroxyalkanoates (PHA) can be accumulated as inclusion bodies by bacteria. The cell division way and morphology have been shown to play an important role in regulating the bacterial growth and PHA storages. RESULTS: The common growth pattern of Escherichia coli was changed to multiple fission patterns by deleting fission related genes minC and minD together, allowing the formation of multiple fission rings (Z-rings) in several positions of an elongated cell, thus a bacterial cell was observed to be divided into more than two daughter cells at same time. To further improve cell growth and PHA production, some genes related with division process including ftsQ, ftsL, ftsW, ftsN and ftsZ, together with the cell shape control gene mreB, were all overexpressed in E. coli JM109 ∆minCD. The changing pattern of E. coli cell growth and morphology resulted in more cell dry weights (CDW) and more than 80 % polyhydroxybutyrate (PHB) accumulation increases compared to its binary fission control grown under the same conditions. CONCLUSIONS: This study clearly demonstrated that combined over-expression genes ftsQ, ftsW, ftsN, ftsL and ftsZ together with shape control gene mreB in multiple division bacterial E. coli JM109 ∆minCD benefited PHA accumulation. Our study provides useful information on increasing the yield of PHA by changing the cell division pattern and cell morphology of E. coli.

Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis.

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is used to edit eukaryotic genomes. Here, we show that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. As a case study, CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition. A pathway was constructed in Escherichia coli for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from glucose. The native gene sad encoding E. coli succinate semi-aldehyde dehydrogenase was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) for regulating carbon flux to 4-hydroxybutyrate (4HB) biosynthesis. The system allowed formation of P(3HB-co-4HB) consisting of 1-9mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes sucC, sucD and sdhA, sdhB) was channeled preferentially to the 4HB precursor by using selected sgRNAs such as sucC2, sucD2, sdhB2 and sdhA1 via CRISPRi. The resulting 4HB content in P(3HB-co-4HB) was found to range from 1.4 to 18.4mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli.

Production of poly(3-hydroxypropionate) and poly(3-hydroxybutyrate-co-3-hydroxypropionate) from glucose by engineering Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial polyhydroxyalkanoates (PHA) synthesized by bacteria grown on 1,3-propandiol or glycerol. In this study synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli was constructed harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyzes formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pdup) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP. When phaA and phaB from Ralstonia eutropha respectively encoding ß-ketothiolase and acetoacetate reductase, were introduced into the above P3HP producing recombinant E. coli, copolymers poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) were synthesized from glucose as a sole carbon source. The above E. coli recombinants grown on glucose LB medium successfully produced 5g/L cell dry weight containing 18% P3HP and 42% P(3HB-co-84mol% 3HP), respectively, in 48h shake flask studies.

A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation.

BACKGROUND: With the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically. RESULTS: The linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3-4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production. CONCLUSION: The linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.

Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates.

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI(q)-Ptrc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4-6 mol% 3-hydroxyvalerate or 3 HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost.