Cloning, Expression, and In Silico Analysis of Class IV Poly-(R)-3- hydroxybutyrate Genes from New Strain of Bacillus thuringiensis TH-01 Zuhdina Sabiqoh, Rukman Hertadi, Enny Ratnaningsih * Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Indonesia 1. Introduction The development of human understanding in nature sustainability increases the need for the use of sustainable materials. Poly-(R)-3-hydroxybutyrate (PHB) as biodegradable thermoplastic is one of sustainable materials that is expected to be a substituent of conventional petroleum-based plastics (Sharma 2019). Various studies revealed that PHB possessed much wider application compared to the scope of petroleum-based plastics (Ray and Kalia 2017). These findings further increase the demand of PHB but seem to have encountered challenges in its production, particularly from bacteria (Markets and Markets 2019, Polyhydroxyalkanoate (PHA) market). Recombinant DNA technology is considered as the most powerful tool to increase the production of various enzymes involved in biomaterials production, including PHB (El Rabey et al. 2017). One of the concerns in this approach was the selection of targeted genes sources. In this case, wild type bacteria that able to produce high amount of PHB are potential candidates to be used as PHB biosynthetic genes sources. Previous report had identified a new strain of Bacillus thuringiensis TH-01 that was accumulate 10.5% (w/w of dry cell) of PHB (Rizki et al. 2021). PHB production could be further improve by cloning the PHB biosynthetic genes from this strain into a particular expression vector regulated under a very strong promoter. In bacteria, PHB biosynthesis begins with the uptake of carbon sources from the environment into the cells. These compounds were then converted into acetyl-CoA through glycolysis and followed by three stages of secondary metabolism involving three different enzymes, known as acetoacetyl- CoA acetyltransferase (PhaA) that catalyzes the condensation of two molecules of acetyl-CoA into acetoacetyl-CoA, acetoacetyl-CoA reductase (PhaB) that catalyzes the reduction of acetoacetyl-CoA to produce (R)-3-hydroxybutyryl-CoA, and PHA synthase that catalyzes the polymerization of (R)-3- hydroxybutyryl-CoA to poly-(R)-3-hydroxybutyrate (Tsuge et al. 2015). PHA synthase is classified into four types of proteins (Chek et al. 2017). Type I which is PhaC, type III that consists of PhaC and PhaE ABSTRACT Poly-(R)-3-hydroxybutyrate (PHB) is a bioplastic derivative of polyhydroxyalkanoate (PHA) which can be synthesized by bacteria under certain growth conditions. Previous study has reported a new strain of Bacillus thuringiensis TH-01 isolated from thermite, which found to accumulate PHB. This research aimed to clone PHB biosynthesis genes from B. thuringiensis TH-01 and study its expression as well as predict the tertiary structure of the enzymes. The clone of phaA gene, which encodes PhaA, was obtained as 1182 bp. On the other hand, 2546 bp clone of phaRBC gene cluster was obtained to consist of 744 bp phaB, 1086 bp phaC, and 483 bp phaR, encoding respective PhaB, PhaC, and PhaR proteins. In silico analysis indicated that PhaA, PhaB, PhaC, and PhaR, revealed to have 393, 247, 361, and 160 amino acid, respectively. The predicted model of PhaA, PhaB, and PhaC showed dominant structure of α/β folding motif, while PhaR was dominated by a helix-loop-helix motif. The catalytic residues of PhaA were Cys88, His349, and Cys379, whereas the catalytic residues of PhaB were Ser142, Tyr155, and Lys159. These catalytic residues were identical to those residues obtained in other PHB biosynthetic enzymes reported elsewhere, confrming that our clones were of PHB biosynthetic genes. ARTICLE INFO Article history: Received September 18, 2021 Received in revised form December 23, 2021 Accepted January 21, 2022 KEYWORDS: Bacillus thuringiensis, phaA gene, phaRBC gene cluster, poly-(R)-3-hydroxybutyrate * Corresponding Author E-mail Address: ennyratnaningsih1958@gmail.com Vol. 29 No. 3, May 2022 310-319 DOI:10.4308/hjb.29.3.310-319 ISSN: 1978-3019 EISSN: 2086-4094 H A Y AT I H A Y AT I Journal of Biosciences Journal of Biosciences