Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli.

Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase-enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. KEY POINTS: • Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. • Improved production of short-chain alcohol derived multimethyl-branched esters. • Establishing ethanologenic pathways for de novo production of ethanol derived MBE. • Characterization of a novel phenylethanol-derived MBE.

High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments.

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

Deteriorating effects of lichen and microbial colonization of carbonate building rocks in the Romanesque churches of Segovia, Spain.

In this study, the deterioration effects of lichens and other lithobionts in a temperate mesothermal climate were explored. We examined samples of dolostone and limestone rocks with visible signs of biodeterioration taken from the exterior wall surfaces of four Romanesque churches in Segovia (Spain): San Lorenzo, San Martín, San Millán and La Vera Cruz. Biofilms developing on the lithic substrate were analyzed by scanning electron microscopy. The most common lichen species found in the samples were recorded. Fungal cultures were then obtained from these carbonate rocks and characterized by sequencing Internal Transcribed Spacers (ITS). Through scanning electron microscopy in back-scattered electron mode, fungi (lichenized and non-lichenized) were observed as the most frequent microorganisms occurring at sites showing signs of biodeterioration. The colonization process was especially conditioned by the porosity characteristics of the stone used in these buildings. While in dolostones, microorganisms mainly occupied spaces comprising the rock's intercrystalline porosity, in bioclastic dolomitized limestones, fungal colonization seemed to be more associated with moldic porosity. Microbial biofilms make close contact with the substrate, and thus probably cause significant deterioration of the underlying materials. We describe the different processes of stone alteration induced by fungal colonization and discuss the implications of these processes for the design of treatments to prevent biodeterioration.