Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability.

The pursuit of carbon neutrality goals has sparked considerable interest in expanding bioplastics production from microbial cell factories. One prominent class of bioplastics, polyhydroxyalkanoates (PHA), is generated by specific microorganisms, serving as carbon and energy storage materials. To begin with, a native PHA producer, Cupriavidus necator (formerly Ralstonia eutropha) is extensively studied, covering essential topics such as carbon source selection, cultivation techniques, and accumulation enhancement strategies. Recently, various hosts including archaea, bacteria, cyanobacteria, yeast, and plants have been explored, stretching the limit of microbial PHA production. This review provides a comprehensive overview of current advancements in PHA bioproduction, spanning from the native to diversified cell factories. Recovery and purification techniques are discussed, and the current status of industrial applications is assessed as a critical milestone for startups. Ultimately, it concludes by addressing contemporary challenges and future prospects, offering insights into the path towards reduced carbon emissions and sustainable development goals.

Heterologous Expression of Toxic White Spot Syndrome Virus (WSSV) Protein in Eengineered Escherichia coli Strains.

Aquacultural shrimps suffer economic lost due to the white spot syndrome virus (WSSV) that is the most notorious virus for its fatality and contagion, leading to a 100% death rate on infected shrimps within 7 days. However, the infection of mechanism remains a mystery and crucial problem. To elucidate the pathogenesis of WSSV, a high abundance of protein is required to identify and characterize its functions. Therefore, the optimal WSSV355 overexpression was explored in engineered Escherichia coli strains, in particular C43(DE3) as a toxic tolerance strain remedied 40% of cell growth from BL21(DE3). Meanwhile, a trace amount of WSSV355 was observed in both strains. To optimize the codon of WSSV355 using codon adaption index (CAI), an overexpression was observed with 1.32 mg/mL in C43(DE3), while the biomass was decreased by 35%. Subsequently, the co-expression with pRARE boosted the target protein up to 1.93 mg/mL. Finally, by scaling up production of WSSV355 in the fermenter with sufficient oxygen supplied, the biomass and total and soluble protein were enhanced 67.6%, 44.9%, and 7.8% compared with that in flask condition. Herein, the current approach provides efficacious solutions to produce toxic proteins via codon usage, strain selection, and processing optimization by alleviating the burden and boosting protein production in E. coli.

High-level production and extraction of C-phycocyanin from cyanobacteria Synechococcus sp. PCC7002 for antioxidation, antibacterial and lead adsorption.

Global warming and climate change because carbon dioxide (CO2) release to atmosphere is the forecasting challenges to human being. We are facing how to overcome the dilemma on the balance between economic and environment, thus taking more efforts on green processes to meet agreement of sustainable society are urgent and crucial. The absorption of CO2 by microalgae reduces the impact of CO2 on the environment. In this study, the CO2 removal efficiency was the highest in the culture of Cyanobacterium Synechococcus sp. PCC7002 (also called blue-green algae), at 2% CO2 to reach a value of 0.86 g-CO2/g-DCW. The main product of PCC7002 is C-phycocyanin (C-PC) which regarding to phycobilisome complex in all cyanobacterial species. A 160% increasing C-PC was achieved in the cultivation under 100 µmol/m2/s light intensity, 12:12 light-period with 2% CO2 at 30 °C. The mix-culture of nitric and ammonia ions had positive effect on the cell growth and C-PC accumulation, thus realized the highest yield of 0.439 g-CPC/g-DCW. Additionally, the partial purified C-PC displayed 89% antioxidant activity of 2,2-diphenyl-1-picryhydrazyl (DPPH) and 11% of superoxide free radical scavenging activity, respectively. The production of C-PC from PCC7002 reduced the CO2 emission and exhibited antibacterial activity against Escherichia coli and lead ion adsorption at room temperature, which has the great potential for eco-friendly application.

Purification and biofabrication of 5-aminolevulinic acid for photodynamic therapy against pathogens and cancer cells.

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid which has involved in heme metabolism of organisms, and has been widely applied in agriculture, and medical fields nowadays. 5-ALA is used in the elimination of pathogens or cancer cells by photodynamic therapy (PDT) owing to the photosensitizer reaction which releases the reactive oxygen species (ROS). Currently, biofabrication of 5-ALA is regarded as the most efficient and eco-friendly approach, but the complicated ingredient of medium causes the nuisance process of purification, resulting in low recovery and high producing cost. In this study, hydrogen chloride, sodium acetate, and ammonia were examined to maximize the recovery of 5-ALA from ion-exchange chromatography (IEC), thus a 92% recovery in 1 M ammonia at pH 9.5 was obtained. Afterward, the activated carbon was used for decolorization to further remove the pigments from the eluent. Four organic solvents, i.e., diethyl ether, methanol, ethanol, and acetone were compared to extract and form 5-ALA precipitation. The purified 5-ALA was verified to eliminate 74% of A549 human lung cancer and 83% of A375 melanoma skin cancer cell. Moreover, Proteus hauseri, Aeromonas hydrophila, Bacillus cereus, and Staphylococcus aureus were killed via anti-microbial PDT with 1% 5-ALA and reached 100% killing rate at optimal condition. With the addition of 0.05% 5-ALA during the culture, the growth of microalgae Chlorella sorokiniana was improved to against a common aquatic pathogen, A. hydrophila. The broad application of 5-ALA was demonstrated in this study for the first time.

Migration of glutamate decarboxylase by cold treatment on whole-cell biocatalyst triggered activity for 4-aminobutyric acid production in engineering Escherichia coli.

Glutamate decarboxylase B (GadB) from Escherichia coli, an intrinsic pyridoxal 5'-phosphate (PLP)-dependent enzyme has been employed for 4-aminobutyric acid (GABA) biosynthesis, which involves the glutamate import and GABA export via a transporter located in the inner membrane as rate determined step of whole-cell (WC) biotransformation. Herein, GadB was cloned and overexpressed in E. coli under a constitutive promoter in a high copy number plasmid, and 46.9 g/L GABA was produced. It was observed that GadB migrated to the periplasm when the WC were subjected to -20 °C cold treatment for 24 h prior to the biotransformation. Kinetic studies indicated that the enzymatic turnover rate of WC increased 2-fold after cold treatment, which was correlated with the migration rate of GadB, and up to 88.6% of GadB. The export or possible migration of GadB mitigated the rate-limiting step of WC biotransformation, and a 100% conversion of substrate to GABA was obtained. Finally, we launched a promising strategy for GABA production of 850 g/L from cost-effective monosodium glutamate (MSG) by using WC biocatalysts with 10-times recycling.

Plasmid-Free System and Modular Design for Efficient 5-Aminolevulinic Acid Production by Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an essential intermediate for many organisms and has been considered for the applications of medical especially in photodynamic therapy of cancer recently. However, ALA production via chemical approach is complicated; hence, microbial manufacturing has received more attentions. In this study, a modular design to simultaneously express ALA synthase from Rhodobacter sphaeroides (RshemA), a non-specific ALA exporter (RhtA), and chaperones was first developed and discussed. The ALA production was significantly increased by coexpressing RhtA and RshemA. Besides, ALA was enhanced by the cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes of pdxK and pdxY or direct addition. However, inclusion bodies of RshemA served as an obstacle; thus, chaperones DnaK and GroELS were introduced to reform the conformation of proteins and successfully improved ALA production. Finally, a plasmid-free strain RrGI, as the robust chassis, was established and a 6.23-fold enhancement on ALA biosynthesis and led to 7.47 g/L titer and 0.588 g/L/h productivity under the optimal cultural condition.

Redirection of metabolic flux in Shewanella oneidensis MR-1 by CRISPRi and modular design for 5-aminolevulinic acid production.

Programming non-canonical organisms is more attractive due to the prospect of high-value chemical production. Among all, Shewanella oneidensis MR-1 possesses outstanding heme synthesis ability and is well-known for electron transfer, thus has high potential in microbial fuel cell and bioremediation. However, heme, as the final product of C4 and C5 pathways, is regulated by heme cluster for the high-value 5-aminolevulinic acid (ALA) for cancer photodynamic therapy, which has never been explored in MR-1. Herein, the heme metabolism in MR-1 was firstly optimized for ALA production. We applied CRISPR interference (CRISPRi) targeted on the genes to fine-tune carbon flux in TCA cycle and redirected the carbon out-flux from heme, leading to a significant change in the amino acid profiles, while downregulation of the essential hemB showed a 2-fold increasing ALA production via the C5 pathway. In contrast, the modular design including of glucokinase, GroELS chaperone, and ALA synthase from Rhodobacter capsulatus enhanced ALA production markedly in the C4 pathway. By integrating gene cluster under dual T7 promoters, we obtained a new strain M::TRG, which significantly improved ALA production by 145-fold. We rewired the metabolic flux of MR-1 through this modular design and successfully produced the high-value ALA compound at the first time.

Challenges and opportunities of bioprocessing 5-aminolevulinic acid using genetic and metabolic engineering: a critical review.

5-Aminolevulinic acid (5-ALA), a non-proteinogenic five-carbon amino acid, has received intensive attentions in medicine due to its approval by the US Food and Drug Administration (FDA) for cancer diagnosis and treatment as photodynamic therapy. As chemical synthesis of 5-ALA performed low yield, complicated processes, and high cost, biosynthesis of 5-ALA via C4 (also called Shemin pathway) and C5 pathway related to heme biosynthesis in microorganism equipped more advantages. In C4 pathway, 5-ALA is derived from condensation of succinyl-CoA and glycine by 5-aminolevulic acid synthase (ALAS) with pyridoxal phosphate (PLP) as co-factor in one-step biotransformation. The C5 pathway involves three enzymes comprising glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (HemA), and glutamate-1-semialdehyde aminotransferase (HemL) from α-ketoglutarate in TCA cycle to 5-ALA and heme. In this review, we describe the recent results of 5-ALA production from different genes and microorganisms via genetic and metabolic engineering approaches. The regulation of different chassis is fine-tuned by applying synthetic biology and boosts 5-ALA production eventually. The purification process, challenges, and opportunities of 5-ALA for industrial applications are also summarized.

Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67 g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66 g/L when 0.1 mM of IPTG was added at early exponential phase (i.e., OD600 was equal to 0.7 to 0.8), while 6 g/L of glycine, 2 g/L of succinate, and 0.03 mM of pyridoxal 5'-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

Mechanism study of photo-induced gold nanoparticles formation by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1, a bioelectricity generating bacterium, is broadly used in bioremediation, microbial fuel cell and dissimilatory reduction and recovery of precious metals. Herein, we report for the first time that photo induction as a trigger to stimulate gold nanoparticles (Au@NPs) formation by MR-1, with wavelength and light intensity as two key variables. Results indicated that sigmoidal model is the best fit for Au@NPs formation at various wavelengths (with R2 > 0.97). Light intensity in terms of photosynthetic photon flux density (PPFD) critically influences the rate constant in the low-light intensity region (PPFD < 20), while wavelength controls the maximum rate constant in the high-light region (PPFD > 20). By deletion of Mtr pathway genes in MR-1, we proposed the mechanism for light induced Au@NP formation is the excitation effect of light on certain active groups and extracellular polymeric substances (EPS) on the cell surface. Also, the release of electrons from proteins and co-enzyme complexes enhance electron generation. To the best of our knowledge, this is the first-attempt to explore the effect of photo-induction on Au@NPs production by MR-1, which provides an alternative cost-effective and eco-friendly process in green chemical industry.

Identification of Gold Sensing Peptide by Integrative Proteomics and a Bacterial Two-Component System.

The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel GBP. Finally, the heterologous over-expression of GBP by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.

Tobacco consumption and behavior of the smokers in Jiangxi province / 中华流行病学杂志

Objective To explore the behaviors of smokers as well as the tobacco consumption in Jiangxi province.Impact regarding the increases of tobacco retail price on the smoking behaviors among cigarette consumers was also studied.Data was provided for further development on measurements including legislation,taxing and increasing the retail price on tobacco.Methods 805 residents above 15 year-olds were chosen through random clustered sampling,and face to face interviewed,in Shangrao and Yichun of Jiangxi province.A chart was drawn to show the relationship between the change of smoking behavior and the increase of sales price of tobacco.Results The average rate of smoking was 27.08％ (46.62％ for males and 3.05％ for females).16.06％ (35/218) of the smokers had ever tried to quit smoking but failed.The years of smoking ranged from 1 year to 50 years,with 96.33％ of the smokers purchased cigarettes on their own.Price of the cigarette ranged from 15 to 30 RMB per package,and smokers usually spent on average 192 RMB which accounted 13％ of their monthly income,per month.According to the chart,if the cigarette price had a 50％ increase,it would lead to 45％ of the smokers reduce the number of cigarette consumption with another 5％ of the smokers attempt to quit smoking.If cigarette price had an increase by 2 times,50％ of the smokers would attempt to quit smoking.So the chart could be used to estimate the percentage of smokers' behavior change according to the increase of tobacco's sales price.Conclusion The cconomy burden of tobacco consumption among cigarette smokers was huge,suggesting that the Chinese smokers did need to change their smoking behavior.Chinese government should develop tobacco-related legislations and laws and set proper cigarette tax,hopefully this could reduce the number of smokers in a long run.

Nitric oxide synthase (NOS) expression and nitric oxide (NO) content in the skeletal muscles in transverse process syndrome of the 3rd lumbar vertebra of model rats treated with acupotomology therapy / 中国骨伤

<p><b>OBJECTIVE</b>To study the relation between the nitric-oxide synthase (NOS) expression and nitric oxide (NO) content in the skeletal muscles and the injury condition of soft tissue in the 3rd lumbar vertebrae syndrome model rats, and to observe the effect of acupotomology therapy.</p><p><b>METHODS</b>One hundred and twenty-eight adult SD rats were allocated to 4 groups randomly: normal group, model group, aminoguanidin group and acupotomology treatment group, 32 rats in each group. NOS expression, NO content and injury of the soft tissue in the 3rd lumbar vertebra were observed on the 1st, 3rd, 7th and 14th day after the acupotomology treatment and aminoguanidine intervention.</p><p><b>RESULTS</b>1) Inducible NOS (iNos) activity and NO content in model group was significantly higher (F = 522.860, P < 0.01), in acupotomology group and aminoguanidine group was significantly lower than the model group (FiNOS = 28.894, P < 0.01), and iNOS activity and NO content in all groups was in competence with the condition of soft tissue injuries. 2) Endothelium NOS (eNOS) expression raised in model group and acupotomology group, and achieve peak on the 7th day. There was significant difference between the eNOS expression in acupotomology group and the model group (FeNOS = 3.454, P < 0.05). 3) The expression of neuron NOS (nNOS) in the model group, aminoguanidine group and acupotomology group had no significant (FnNOS = 0.962, P > 0.05).</p><p><b>CONCLUSION</b>Acupotomology treatment can restrain the development of high content NO, release the inflammatory reaction and injury condition, improve microcirculation, prevent the development of scar tissue of the injured soft tissue, and has significant recovering effectiveness in the soft tissue injured model rats.</p>