Biofuel Research Journal

Biofuel Research Journal

Targeted silencing of exopolysaccharide biosynthesis genes via CRISPR/dCas9 to enhance polyhydroxybutyrate production from biodiesel-derived crude glycerol in Burkholderia sp.

Document Type : Research Paper

Authors
Kanokjun Jaiboon 1
Kentaro Miyazaki 2
Kohsuke Honda 2
Suchada Chanprateep Napathorn 1, 2, 3
1 Program in Biotechnology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
2 International Center for Biotechnology, The University of Osaka, Suita, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
3 Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
10.18331/BRJ2025.12.4.3
Abstract
The newly isolated Burkholderia sp. SCN-KJ, which is capable of simultaneously producing intracellular polyhydroxybutyrate (PHB) and exopolysaccharide (EPS) from biodiesel-derived crude glycerol, presented a significant challenge in this study because of interference from EPS synthesis. The bceA gene, which encodes a bifunctional protein responsible for mannose-6-phosphate isomerization and the mannose-1-phosphate guanylyltransferase reaction, was identified as a key regulator of EPS production. To address this issue, CRISPR interference (CRISPRi) was employed to target the bceA gene, leading to the complete suppression of EPS synthesis and a significant improvement in PHB yield. Under optimized culture conditions, the ΔbceA mutant strain exhibited complete inhibition of EPS production, resulting in a 1.75-fold increase in the PHB concentration and PHB of a higher molecular weight than that of the wild-type Burkholderia sp. SCN-KJ. These results highlight the effectiveness of dCas9-mediated silencing of the bceA gene in overcoming the challenges posed by EPS interference and underscore its potential for increasing PHB production in Burkholderia sp. SCN-KJ.

Graphical Abstract

Targeted silencing of exopolysaccharide biosynthesis genes via CRISPR/dCas9 to enhance polyhydroxybutyrate production from biodiesel-derived crude glycerol in Burkholderia sp.

Highlights

  • CRISPRi-mediated silencing of bceA suppressed EPS synthesis in Burkholderia sp. SCN-KJ.
  • A novel Burkholderia sp. SCN-KJ and its ΔbceA mutant valorized biodiesel-derived crude glycerol to PHB.
  • The ΔbceA mutant produced 4.9 ± 0.1 g/L PHB with 65.7 ± 0.1% (w/w) PHB content.
  • EPS deficiency in ΔbceA validated the essential role of bceA in EPS biosynthesis.
  • The ΔbceA mutant increased PHB production by 1.75-fold and increased the Mw.

Keywords
Burkholderia sp
CRISPRi
Gene silencing
Polyhydroxybutyrate
Green production
Life cycle assessment
[1] Alvarez Chavez, B., Raghavan, V., Tartakovsky, B., 2022. A comparative analysis of biopolymer production by microbial and bioelectrochemical technologies. RSC Adv 12(25), 16105-16118.
[2] Arshad, Z., Shin, K.H., Bae, H., Hur, J., 2022. Using a stable isotope tracing technique to elucidate the effect of substrate C/N ratio on the formation of different constituents of extracellular polymeric substances in an aerobic-anoxic sequencing batch reactor. J. Water Process Eng. 50, 103262.
[3] Ashby, R.D., Solaiman, D.K.Y., Nuñez, A., Strahan, G.D., Johnston, D.B., 2018. Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid. Bioresour Technol 253, 333-342.
[4] Assal, N., Lin, M., 2021. PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis. J. Microbiol. Methods. 181, 106121.
[5] Barham, P.J., Keller, A., Otun, E.L., Holmes, P.A., 1984. Crystallization and morphology of a bacterial thermoplastic: poly-3-hydroxybutyrate. J. Mater. Sci. 19(9), 2781-2794.
[6] Bartholdson, S.J., Brown, A.R., Mewburn, B.R., Clarke, D.J., Fry, S.C., Campopiano, D.J., Govan, J.R.W., 2008. Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex. Microbiology. 154(8), 2513-2521.
[7] BBGI Public Company Limited, 2024. Biodiesel business.
[8] BD, 2021. BD Acquires Tepha, Inc., to Drive New Innovations in Soft Tissue Repair and Regeneration.
[9] Boontip, T., Waditee-Sirisattha, R., Honda, K., Napathorn, S.C., 2021. Strategies for poly(3-hydroxybutyrate) production using a cold-shock promoter in Escherichia coli. Frontiers in Bioeng. Biotechnol. 9.
[10] Borlee, G.I., Plumley, B.A., Martin, K.H., Somprasong, N., Mangalea, M.R., Islam, M.N., Burtnick, M.N., Brett, P.J., Steinmetz, I., AuCoin,
D.P., Belisle, J.T., Crick, D.C., Schweizer, H.P., Borlee, B.R., 2017. Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster. PLOS Negl. Trop. Dis. 11(6), e0005689.
[11] Borrero-de Acuña, J.M., Bielecka, A., Häussler, S., Schobert, M., Jahn, M., Wittmann, C., Jahn, D., Poblete-Castro, I., 2014. Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida. Microb. Cell Fact. 13(1), 88.
[12] Bourles, A., Amir, H., Gensous, S., Cussonneau, F., Medevielle, V., Juillot, F., Bazire, A., Meyer, M., Burtet-Sarramegna, V., Cavaloc, Y., Jourand, P., Guentas, L., 2020. Investigating some mechanisms underlying stress metal adaptations of two Burkholderia sensu lato species isolated from New Caledonian ultramafic soils. Eur. J. Soil Biol. 97, 103166.
[13] Bourles, A., Pierre, G., Amir, H., Le Floc’h, A., Chalkiadakis, E., Médevielle, V., Jourand, P., Michaud, P., Burtet-Sarramégna, V., Guentas, L., 2024. The cepacian-like exopolysaccharide of Paraburkholderia ultramafica STM10279T enhances growth and metal adaptation of Tetraria comosa on New Caledonian ultramafic soil. Front. Plant Sci. Volume. 15.
[14] Carlota, V., 2019. Genecis is converting food waste into 3D printing filaments.
[15] Carlson, B., 2007. 'Natural plastics' ripe with potential. Biotechnol Healthc 4(3), 11-18.
[16] Castellane, T.C.L., Persona, M.R., Campanharo, J.C., de Macedo Lemos, E.G., 2015. Production of exopolysaccharide from rhizobia with potential biotechnological and bioremediation applications. Int. J. Biol. Macromol. 74, 515-522.
[17] Cescutti, P., Foschiatti, M., Furlanis, L., Lagatolla, C., Rizzo, R., 2010. Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by bacteria of the Burkholderia cepacia complex. Carbohydr. Res. 345(10), 1455-1460.
[18] Chen, E., Yang, C., Tao, W., Li, S., 2024. Polysaccharides produced by plant growth-promoting rhizobacteria strain Burkholderia sp. BK01 enhance salt stress tolerance to Arabidopsis thaliana. Polymers. 16(1), 145.
[19] Chen, F., Ye, J., Liu, W., Chio, C., Wang, W., Qin, W., 2021. Knockout of a highly GC-rich gene in Burkholderia pyrrocinia by recombineering with freeze-thawing transformation. Mol. Plant Pathol. 22(7), 843-857.
[20] Corsi, G.I., Qu, K., Alkan, F., Pan, X., Luo, Y., Gorodkin, J., 2022. CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context. Nat. Commun. 13(1), 3006.
[21] Cui, Y.W., Gong, X.Y., Shi, Y.P., Wang, Z., 2017. Salinity effect on production of PHA and EPS by Haloferax mediterranei. RSC Adv. 7(84), 53587-53595.
[22] Cuzzi, B., Herasimenka, Y., Silipo, A., Lanzetta, R., Liut, G., Rizzo, R., Cescutti, P., 2014. Versatility of the Burkholderia cepacia complex for the biosynthesis of exopolysaccharides: a comparative structural investigation. PLOS ONE. 9(4), e94372.
[23] Dacosta, C.F., Posada, J.A., Ramirez, A., 2015. Large scale production of polyhydroxyalkanoates (PHAs) from wastewater: a study of techno-economics, energy use and greenhouse gas emissions. Int J Environ Chem Ecol Geol Geophys Eng 9(5), 433-438.
[24] Darzins, A., Frantz, B., Vanags, R.I., Chakrabarty, A.M., 1986. Nucleotide sequence analysis of the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa and comparison with the corresponding Escherichia coli gene manA. Gene. 42(3), 293-302.
[25] Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H.W., Listgarten, J., Root, D.E., 2016. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34(2), 184-191.
[26] Feijoo, P., Marín, A., Sánchez-Safont, E., Tena-Medialdea, J., García-March, J.R., Gámez-Pérez, J., Cabedo, L., 2025. Marine degradation of plastics in Western Mediterranean Sea: comparison between biodegradable and conventional polymers. Polym. Degrad. Stab. 234, 111222.
[27] Ferreira, A.S., Leitão, J.H., Silva, I.N., Pinheiro, P.F., Sousa, S.A., Ramos, C.G., Moreira, L.M., 2010. Distribution of Cepacian biosynthesis genes among environmental and clinical Burkholderia strains and role of cepacian exopolysaccharide in resistance to stress conditions. Appl. Environ. Microbiol. 76(2), 441-450.
[28] Ferreira, A.S., Leitão, J.H., Sousa, S.A., Cosme, A.M., Sá-Correia, I., Moreira, L.M., 2007. Functional analysis of Burkholderia cepacia genes bceD and bceF, encoding a phosphotyrosine phosphatase and a tyrosine autokinase, respectively: role in exopolysaccharide biosynthesis and biofilm formation. Appl. Environ. Microbiol. 73(2), 524-534.
[29] Ferreira, A.S., Silva, I.N., Oliveira, V.H., Cunha, R., Moreira, L.M., 2011. Insights into the role of extracellular polysaccharides in Burkholderia adaptation to different environments. Front. Cell. Infect. Microbiol. 1, 16.
[30] Ferreira, M.R., Queiroga, V., Moreira, L.M., 2024. Genomic editing in Burkholderia multivorans by CRISPR/Cas9. Appl Environ Microbiol 90(2), e0225023.
[31] Fu, B., Yan, Q., 2023. Exopolysaccharide is required for motility, stress tolerance, and plant colonization by the endophytic bacterium Paraburkholderia phytofirmans PsJN. Front. Microbiol. 14.
[32] García-Depraect, O., Lebrero, R., Rodriguez-Vega, S., Bordel, S., Santos-Beneit, F., Martínez-Mendoza, L.J., Aragão Börner, R., Börner, T., Muñoz, R., 2022. Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: kinetics, carbon fate and particle size effect. Bioresour. Technol. 344(Part B), 126265.
[33] Goswami, L., Kushwaha, A., Napathorn, S.C., Kim, B.S., 2023. Valorization of organic wastes using bioreactors for polyhydroxyalkanoate production: recent advancement, sustainable approaches, challenges, and future perspectives. Int. J. Biol. Macromol. 247, 125743.
[34] Han, G., Yoon, J., Lee, C., Lee, E., Yoon, K., Kwak, H.W., Jin, H.J., 2023. Biodegradation behavior of amorphous polyhydroxyalkanoate-incorporated poly(l-lactic acid) under modulated home-composting conditions. Polym. Test. 126, 108155.
[35] Harding, K.G., Dennis, J., Von Blottnitz, H., Harrison, S.T., 2007. Environmental analysis of plastic production processes: comparing petroleum-based polypropylene and polyethylene with biologically-based poly-β-hydroxybutyric acid using life cycle analysis. J. Biotechnol. 130(1), 57-66.
[36] Hogan, A.M., Jeffers, K.R., Palacios, A., Cardona, S.T., 2021. Improved dynamic range of a rhamnose-inducible promoter for gene expression in Burkholderia spp. Appl. Environ. Microbiol. 87(18), e0064721.
[37] Hogan, A.M., Rahman, A., Lightly, T.J., Cardona, S.T., 2019. A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia. ACS Synth. Biol. 8(10), 2372-2384.
[38] Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao, G., Zhang, F., 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31(9), 827-832.
[39] Insulkar, P., Kerkar, S., Lele, S.S., 2018. Purification and structural-functional characterization of an exopolysaccharide from Bacillus licheniformis PASS26 with in vitro antitumor and wound healing activities. Int. J. Biol. Macromol. 120(Part B), 1441-1450.
[40] Jaiboon, K., Chouwatat, P., Napathorn, S.C., 2024. Valorization of biodiesel-derived crude glycerol for simultaneous biosynthesis of biodegradable polyhydroxybutyrate and exopolysaccharide by the newly isolated Burkholderia sp. SCN-KJ. Int. J. Biol. Macromol. 281(Part 4), 136556.
[41] Jiang, G., Gan, L., Li, X., He, J., Zhang, S., Chen, J., Zhang, R., Xu, Z., Tian, Y., 2021. Characterization of structural and physicoechemical properties of an exopolysaccharide produced by Enterococcus sp. F2 from fermented soya beans. Front. Microbiol. 12, 744007.
[42] Jiang, T., Tan, T., Zong, Z., Fan, D., Wang, J., Qiu, Y., Teng, X., Zhang, H.M., Rao, C., 2025. Enhancing oil feedstock utilization for high-yield low-carbon polyhydroxyalkanoates industrial bioproduction. Metab Eng 91, 44-58.
[43] Jyoti, K., Soni, K., Chandra, R., 2024. Optimization of the production of Exopolysaccharide (EPS) from biofilm-forming bacterial consortium using different parameters. The Microbe. 4, 100117.
[44] Kang, Y., Norris, M.H., Wilcox, B.A., Tuanyok, A., Keim, P.S., Hoang, T.T., 2011. Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. Nat. Protoc. 6(8), 1085-1104.
[45] Kendall, A., 2012. A life cycle assessment of biopolymer production from material recovery facility residuals. Resour Conserv Recycl 61, 69-74.
[46] Kim, J., Yun, H., Won, S., Lee, D., Baek, S., Heo, G., Park, S., Jin, H.-J., Kwak, H.W., 2024. Comparative degradation behavior of polybutylene succinate (PBS), used PBS, and PBS/Polyhydroxyalkanoates (PHA) blend fibers in compost and marine-sediment interfaces. Sustainable Mater. Technol. 41, e01065.
[47] Kim, S., Dale, B., 2005. Life cycle assessment study of biopolymers (Polyhydroxyalkanoates)-Derived from No-Tilled Corn (11 pp). Int. J. Life Cycle Assess. 10, 200-210.
[48] Koller, M., Mukherjee, A., 2022. A New Wave of Industrialization of PHA Biopolyesters. Bioengineering (Basel) 9(2), 74.
[49] Kozaeva, E., Volkova, S., Matos, M.R.A., Mezzina, M.P., Wulff, T., Volke, D.C., Nielsen, L.K., Nikel, P.I., 2021. Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida. Metab Eng 67, 373-386.
[50] Laird, K., 2023. Bluepha launches commercial PHA portfolio.
[51] Lardi, M., Aguilar, C., Pedrioli, A., Omasits, U., Suppiger, A., Cárcamo-Oyarce, G., Schmid, N., Ahrens, C.H., Eberl, L., Pessi, G., 2015. σ54-Dependent response to nitrogen limitation and virulence in Burkholderia cenocepacia strain H111. Appl. Environ. Microbiol. 81(12), 4077-4089.
[52] Lee, W., Kim, J., Lee, T.G., 2025. Certifications and testing methods for biodegradable plastics. Rev. Chem. Eng. 41(2), 125-146.
[53] Li, D., Lv, L., Chen, J.-C., Chen, G.-Q., 2017. Controlling microbial PHB synthesis via CRISPRi. Appl Microbiol Biotechnol 101(14), 5861-5867.
[54] Lin, L., Chen, J., Mitra, R., Gao, Q., Cheng, F., Xu, T., Zuo, Z., Xiang, H., Han, J., 2021. Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux. Commun Biol 4(1), 1007.
[55] Lin, Y.-C., Diankristanti, P.A., Chen, P.-Y., Yi, Y.-C., Ng, I.S., 2025. Sustainable Polyhydroxybutyrate Production via Metabolic Flux Redirection Using Clustered Regularly Interspaced Short Palindromic Repeats Interference in Escherichia coli and Carbon Capture with Microalgae. ACS Sustain Chem Eng 13(11), 4418-4426.
[56] Liu, Y., Bellich, B., Hug, S., Eberl, L., Cescutti, P., Pessi, G., 2020. The exopolysaccharide cepacian plays a role in the establishment of the Paraburkholderia phymatum - Phaseolus vulgaris symbiosis. Front. Microbiol. 11.
[57] Liu, Y., Lardi, M., Pedrioli, A., Eberl, L., Pessi, G., 2017. NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111. PLOS ONE. 12(6), e0180362.
[58] Long, J., Zeng, Y., Liang, F., Liu, N., Xi, Y., Sun, Y., Zhao, X., 2024. Transformed Salmonella typhimurium SL7207/pcDNA-CCOL2A1 as an orally administered DNA vaccine. AMB Expr. 14(1), 6.
[59] Lopez-Arenas, T., González-Contreras, M., Anaya-Reza, O., Sales-Cruz, M., 2017. Analysis of the fermentation strategy and its impact on the economics of the production process of PHB (polyhydroxybutyrate). Comput. Chem. Eng. 107, 140-150.
[60] Majerczak, K., Liggat, J.J., 2024. Submission to journal of polymers and the environment evaluation of thermal properties and crystallinity in PHB-based systems-A DoE approach. J. Polym. Environ. 32(9), 4613-4632.
[61] Masłowska-Górnicz, A., van den Bosch, M.R.M., Saccenti, E., Suarez-Diez, M., 2022. A large-scale analysis of codon usage bias in 4868 bacterial genomes shows association of codon adaptation index with GC content, protein functional domains and bacterial phenotypes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1865(6), 194826.
[62] McAdam, B., Brennan Fournet, M., McDonald, P., Mojicevic, M., 2020. Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polymers. 12(12), 2908.
[63] Mohamad, N.I., Harun, A., Hasan, H., Deris, Z.Z., 2022. In-vitro activity of β-lactams/trimethoprim-sulfamethoxazole combinations against different strains of Burkholderia pseudomallei. Trop. Biomed. 39(1), 11-16.
[64] Morcillo, R.J.L., Manzanera, M., 2021. The effects of plant-associated bacterial exopolysaccharides on plant abiotic stress tolerance. Metabolites. 11(6), 337.
[65] Muangwong, A., Boontip, T., Pachimsawat, J., Napathorn, S.C., 2016. Medium chain length polyhydroxyalkanoates consisting primarily of unsaturated 3-hydroxy-5-cis-dodecanoate synthesized by newly isolated bacteria using crude glycerol. Microb. Cell Fact. 15(55), 1-17.
[66] Napathorn, S.C., Visetkoop, S., Pinyakong, O., Okano, K., Honda, K., 2021. Polyhydroxybutyrate (PHB) production using an Arabinose-Inducible expression system in comparison with cold shock inducible expression system in Escherichia coli. Front. Bioeng. Biotechnol. 9, 661096.
[67] Narancic, T., Verstichel, S., Reddy Chaganti, S., Morales-Gamez, L., Kenny, S.T., De Wilde, B., Babu Padamati, R., O’Connor, K.E., 2018. Biodegradable plastic blends create new possibilities for end-of-life management of plastics but they are not a panacea for plastic pollution. Environ. Sci. Technol. 52(18), 10441-10452.
[68] Ogawa, R., 2020. Marine-degradable plastic progressing for popularization under new international standards. Mitsui & Co. Global Strategic Studies Institute,
[69] Pal, G., Saxena, S., Kumar, K., Verma, A., Sahu, P.K., Pandey, A., White, J.F., Verma, S.K., 2022. Endophytic Burkholderia: multifunctional roles in plant growth promotion and stress tolerance. Microbiol. Res. 265, 127201.
[70] Paul, S., Parvez, S.S., Goswami, A., Banik, A., 2024. Exopolysaccharides from agriculturally important microorganisms: conferring soil nutrient status and plant health. Int. J. Biol. Macromol. 262(Part 2), 129954.
[71] Phothong, N., Boontip, T., Chouwatat, P., Aht-Ong, D., Napathorn, S.C., 2024. Preparation and characterization of astaxanthin-loaded biodegradable polyhydroxybutyrate (PHB) microbeads for personal care and cosmetic applications. Int. J. Biol Macromol. 257(Part 2), 128709.
[72] Prasad, J.K., Dey, R., Raghuwanshi, R., 2022. Exopolysaccharide-Producing Rhizospheric Bacteria Enhance Yield via Promoting Wheat (Triticum aestivum L.) Growth at Early Stages. Microbiology. 91(6), 757-769.
[73] Prihatna, C., Yan, Q., 2024. Exopolysaccharide is required by Paraburkholderia phytofirmans PsJN to confer drought-stress tolerance in pea. Front. Microbiol. Volume. 15.
[74] Raunhan, R., Jantharadej, K., Mhuantong, W., Napathorn, S.C., Suwannasilp, B.B., 2023. Valorization of food waste derived anaerobic digestate into polyhydroxyalkanoate (PHA) using Thauera mechernichensis TL1. Waste Manage. 171, 248-258.
[75] Riahi Rad, Z., Riahi Rad, Z., Goudarzi, H., Goudarzi, M., Mahmoudi, M., Yasbolaghi Sharahi, J., Hashemi, A., 2021. MicroRNAs in the interaction between host-bacterial pathogens: a new perspective. J. Cell. Physiol. 236(9), 6249-6270.
[76] Saratale, R.G., Cho, S.K., Bharagava, R.N., Patel, A.K., Vivekanand, V., Bhatia, S.K., Ferreira, L.F.R., Shin, H.S., Awasthi, M.K., Chakrabortty, S., Kumar, R., Saratale, G.D., 2024. Third-generation biomass for bioplastics: a comprehensive review of microalgae-driven polyhydroxyalkanoate production. Biofuel Res. J. 11(4), 2256-2282.
[77] Schilling, C., Ciccone, R., Sieber, V., Schmid, J., 2020. Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365. Metab. Eng. 61, 381-388.
[78] Sousa, S.A., Feliciano, J.R., Pinheiro, P.F., Leitão, J.H., 2013. Biochemical and functional studies on the Burkholderia cepacia complex bceN gene, encoding a GDP-D-mannose 4,6-dehydratase. PLOS ONE. 8(2), e56902.
[79] Sousa, S.A., Moreira, L.M., Leitão, J.H., 2008. Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities. Appl. Microbiol. Biotechnol. 80(6), 1015-1022.
[80] Sousa, S.A., Moreira, L.M., Wopperer, J., Eberl, L., Sá-Correia, I., Leitão, J.H., 2007. The Burkholderia cepacia bceA gene encodes a protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities. Biochem. Biophys. Res. Commun. 353(1), 200-206.
[81] Steiner, E., Shilling, R.E., Richter, A.M., Schmid, N., Fazli, M., Kaever, V., Jenal, U., Tolker-Nielsen, T., Eberl, L., 2022. The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB. npj Biofilms Microbiomes. 8(1), 93.
[82] Thanahiranya, P., Vacharanukrauh, T., Charoensuppanimit, P., Chuetor, S., Chanthanumataporn, M., Assabumrungrat, S., 2025. Techno-economic and environmental assessment of glycerol valorization to polyhydroxybutyrate using chemical digestion methods. Process Saf Environ Prot 195, 106832.
[83] Tsuge, T., 2016. Fundamental factors determining the molecular weight of polyhydroxyalkanoate during biosynthesis. Polym. J. 48(11), 1051-1057.
[84] Uhliariková, I., Matulová, M., Capek, P., 2021. Optimizing acid hydrolysis for monosaccharide compositional analysis of Nostoc cf. linckia acidic exopolysaccharide. Carbohydr. Res. 508, 108400.
[85] Unrean, P., Napathorn, S.C., Tee, K.L., Wong, T.S., Champreda, V., 2023. Lignin to polyhydroxyalkanoate bioprocessing by novel strain of Pseudomonas monteilii. Biomass Convers. Biorefin. 13(6), 4651-4657.
[86] Wang, C., Sheng, W., Zhou, Y., Hang, X., Zhao, J., Gu, Y., Meng, X., Bai, Y., Li, W., Zhang, Y., Zhang, L., Yu, J., Zhou, Z., Li, X., Sun, H., Xue, Y., Xu, T., Zen, K., Ling, H., Zhang, C.Y., Bi, H., Wang, H., 2025. siRNA-AGO2 complex inhibits bacterial gene translation: a promising therapeutic strategy for superbug infection. Cell Rep. Med. 6(3), 101997.
[87] Webb, N.A., Mulichak, A.M., Lam, J.S., Rocchetta, H.L., Garavito, R.M., 2004. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. Protein Sci. 13(2), 529-539.
[88] Wongmoon, C., Napathorn, S.C., 2022. Optimization for the efficient recovery of poly (3-hydroxybutyrate) using the green solvent 1, 3-dioxolane. Front. Bioeng. Biotechnol. 10.
[89] Wu, X.L., Liu, X.W., Wang, Y., Guo, M.Y., Ye, J.R., 2023. Optimization of constitutive promoters using a promoter-trapping vector in Burkholderia pyrrocinia JK-SH007. Int. J. Mol. Sci. 24(11), 9419.
[90] Yang, M., Chen, L., Wang, J., Msigwa, G., Osman, A.I., Fawzy, S., Rooney, D.W., Yap, P.S., 2023. Circular economy strategies for combating climate change and other environmental issues. Environ. Chem. Lett. 21(1), 55-80.
[91] Zhang, R., Xu, W., Shao, S., Wang, Q., 2021. Gene silencing through CRISPR interference in bacteria: current advances and future prospects. Front. Microbiol. 12, 635227.
[92] Zhao, D., Cai, L., Wu, J., Li, M., Liu, H., Han, J., Zhou, J., Xiang, H., 2013. Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei. Appl. Microbiol. Biotechnol. 97(7), 3027-3036.
[93] Zisu, B., Shah, N.P., 2003. Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275. J. Dairy Sci. 86(11), 3405-3415.
[94] Zytner, P., Kumar, D., Elsayed, A., Mohanty, A., Ramarao, B.V., Misra, M., 2023. A review on polyhydroxyalkanoate (PHA) production through the use of lignocellulosic biomass. RSC Sustainability. 1(9), 2120-2134.
Biofuel Research Journal
Volume 12, Issue 4 - Serial Number 4
Autumn 2025
Pages 2537-2553