Biotechnological applications of bacterial microcompartments

Bakterijski mikrorazdelki so proteinski organeli, ki se samo-sestavijo in vključujejo encime, ki izvajajo specifične metabolne procese. Bakterija Salmonella enterica nosi zapis za ene večjih mikrorazdelkov, ki so zapisani na operonu Pdu in so udeleženi pri razgradnji 1,2-propandiola. Mikrorazdelek Pdu gradijo podenote Pdu A, B, B\u27, J, K, N, T, U, ki se najprej samo-sestavijo v homoheksamere oziroma homopentamere, šele nato lahko postanejo gradniki organela. V mikrorazdelek se ponavadi tekom njegove izgradnje vključijo encimi, ki nosijo kratke peptidne značke, s katerimi interagirajo s podenotami organela. Če katero izmed teh peptidnih sekvenc združimo s poljubnim proteinom, lahko dosežemo njegovo pakiranje v mikrorazdelek. Prav zaradi te možnosti so mikrorazdelki biotehnološko zanimivi. Ob zapakiranju encimov določene metabolne poti, lahko dosežemo večjo produktivnost za tarčni produkt, npr. za etanol, ali pa zapakiramo encime metabolne poti, kjer so vmesni intermediati toksični za celico. Zaradi selektivne prepustnosti so mikrorazdelki primerni tudi za sintezo toksičnih proteinov, saj ti ob zapakiranju v organel, ne morejo prosto prehajati po celici. Zavoljo razvoja metode za analizo interakcij protein-DNA smo v laboratoriju v kapside Pdu uspeli zapakirati transkripcijski faktor LacI, ki je imel na amino-terminalnem koncu kratko peptidno sekvenco proteina PduD in na karboksi-terminalnem koncu izboljšani fluorescenčni protein (EGFP). Zasnova eksperimenta omogoča, da celotne mikrorazdelke izoliramo z afinitetno kromatografijo.Bacterial microcompartments are protein organeles involved in various processes in cells. One of the largest microcompartments are found in Salmonella enterica, which are synthesized from the Pdu operon and are involved in 1,2-propandiol metabolism. This microcompartment is composed of subunits: Pdu A, B, B\u27, J, K, N, T, U, which first self-assemble into homohexamers or homopentamers and then assemble into microcompartment. There are several enzymes that are selectively encapsulated in microcompartments via their special peptide carried by the enzyme. This microcompartment packaging sequences can be linked to the protein of interest to direct it into the microcompartment. Due to this property microcompartments have a potential to be exploited as reactors in biotechnology. Researchers encapsulated enzymes from a certain metabolic pathway to obtain larger yield for a target metabolite or they showed that enzymes can be encapsulated, which intermediates or end products are toxic. As similarly to the lipid membrane, also the Pdu protein cages are selective for certain type of metabolites, this precludes that the toxic metabolites are preserved inside the compartments and do not harm the producing cell. In the process of development of the novel method to elucidate nucleoprotein complexes we in this study encapsulated into the Pdu microcompartment a transcription factor LacI, which is fused to short peptide sequence of protein PduD, directing the protein into the compartment, and to enhanced green fluorescent protein (EGFP) at the amino- or the carboxy- terminus, respectively. The experimental setup enabled us to isolate the microcompartments by the affinity cromatography from E. coli cells