A highly robust and productive self-sufficient heterogeneous biocatalysts to asymmetrically reduce β-ketoesters.

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Simvastatin is a top-selling cholesterol-lowering drug traditionally obtained through a semi-synthetic process starting from lovastatin. However, this process is cost-demanding and makes use of chemical reagents that can generate considerable waste. The sustainability concerns underlying the current semisynthetic process encouraged us to immobilize the engineered acyltransferase LovD−BuCh2 on different pore carriers to develop an innovative and sustainable process for the continuous biomanufacturing of simvastatin. We systematically assessed the effect of the immobilization on the functional and structural properties of the immobilized enzyme, the enzyme spatial distribution across the solid material, and the thermal stability of the immobilized biocatalysts. After screening several immobilization carriers, we selected LovD−BuCh2 immobilized on the controlled porous glass particles functionalized with Fe 3+ − catechol complexes as the most suitable heterogeneous biocatalyst to optimize the continuous synthesis of simvastatin. This immobilized enzyme was four times more thermally stable than its free counterpart and maintained >60% product yield during five operational cycles in the batch. Under the optimal flow conditions, we achieved 100% of simvastatin yield with a space-time yield of 4.61 g L −1 h −1 and a specific productivity of 17 mg product × mg enzyme −1 L −1 . This flow-biocatalysis system improves some green chemistry metrics such as the reaction mass efficiency and the atom economy when compared to previous studies of simvastatin manufacturing.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus, but only in few strains of the species Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, in the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

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Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long‐ and short‐massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long‐reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR‐Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high‐quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.