In Silico Design of phaCAB Expression Constructs for Cellulolytic Hosts Toward Hemp Hurd Valorisation and Polyhydroxybutyrate Biosynthesis

Hemp hurds (HHs), an underutilised lignocellulosic biomass (LB) from agricultural waste, offer potential for bioconversion into high-value bioproducts within a circular bioeconomy. Building on prior work involving magnetic nanoparticle-immobilised cellulase hydrolysis of pretreated HH, this study computationally designed candidate phaCAB expression constructs for cellulolytic hosts toward future polyhdroxybutyrate (PHB) production. The objective was to evaluate, in silico, the feasibility of introducing the phaC1, phaA and phaB1 genes from Cupriavidus necator H16 (C. necator) (assembly GCA_000009285.2; loci H16_A1437–H16_A1439) into the cellulolytic hosts Clostridium thermocellum DSM 1313 (C. thermocellum) and Trichoderma reesei RUT C-30 (T. reesei). Coding sequences were retrieved and translated individually and host-specific expression compatibility was assessed via codon adaptation index, effective number of codons, GC content and rare-codon frequency/clustering. Host-specific architectures were designed: three independent tef1-promoter cassettes with fungal Kozak contexts and cbh1 terminators for T. reesei (TrePHB3 integration construct), and a single groEL-promoter operon with graded ribosome-binding sites for the pIKM1-based C. thermocellum construct (pCtPHB1); Escherichia coli BL21 (DE3) (E. coli)/pET-24a(+) served only as an intermediate assembly platform. Clustal Omega alignment, virtual plasmid assembly and simulated restriction digestion (SnapGene) confirmed preservation of the open reading frames and expected fragment sizes. ProtParam analysis indicated instability indices below 40 and negative GRAVY values for PhaC, PhaA and PhaB, indicating overall hydrophilic character. This computational framework links HH valorisation, cellulolytic Consolidated Bioprocessing (CBP) hosts and PHB pathway design within a conceptual biorefinery, supporting future integrated biomass-valorisation platforms; it is computational only and does not demonstrate transformation, expression, PHB accumulation or biomass conversion, which require experimental validation.