Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis.

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product ß-ionone was produced at a titer of 0.87 mg/L by expressing the ß-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

Exogenous glucosylglycerol and proline extend the chronological lifespan of Rhodosporidium toruloides / El glucosilglicerol y la prolina exógenos extienden la vida útil cronológica de Rhodosporidium toruloides

Glucosylglycerol (GG) is an osmolyte found in a few bacteria (e.g., cyanobacteria) and plants grown in harsh environments. GG protects microbes and plants from salinity and desiccation stress. In the industry, GG is synthesized from a combination of ADP-glucose and glycerol-3-phosphate in a condensation reaction catalyzed by glucosylglycerol phosphate synthase. Proline, on the other hand, is an amino acid-based osmolyte that plays a key role in cellular reprograming. It functions as a protectant and a scavenger of reactive oxygen species. Studies on lifespan extension have focused on the use of Saccharomyces cerevisiae. Rhodosporidium toruloides, also known as Rhodotorula toruloides, is a basidiomycetous oleaginous yeast known to accumulate lipids to more than 70% of its dry cell weight. The oleaginous red yeast (R. toruloides) has not been intensely studied in the lifespan domain. We designed this work to investigate how GG and proline promote the longevity of this red yeast strain. The results obtained in our study confirmed that these molecules increased R. toruloides’ viability, survival percentage, and lifespan upon supplementation. GG exerts the most promising effects at a relatively high concentration (100 mM), while proline functions best at a low level (2 mM). Elucidation of the processes underlying these favorable responses revealed that GG promotes the yeast chronological lifespan (CLS) through increased catalase activity, modulation of the culture medium pH, a rise in ATP, and an increase in reactive oxygen species (ROS) accumulation (mitohormesis). It is critical to understand the mechanisms of these geroprotector molecules, particularly GG, and the proclivity of its lifespan application; this will aid in offering clarity on its potential application in aging research.(AU)

Exogenous glucosylglycerol and proline extend the chronological lifespan of Rhodosporidium toruloides.

Glucosylglycerol (GG) is an osmolyte found in a few bacteria (e.g., cyanobacteria) and plants grown in harsh environments. GG protects microbes and plants from salinity and desiccation stress. In the industry, GG is synthesized from a combination of ADP-glucose and glycerol-3-phosphate in a condensation reaction catalyzed by glucosylglycerol phosphate synthase. Proline, on the other hand, is an amino acid-based osmolyte that plays a key role in cellular reprograming. It functions as a protectant and a scavenger of reactive oxygen species. Studies on lifespan extension have focused on the use of Saccharomyces cerevisiae. Rhodosporidium toruloides, also known as Rhodotorula toruloides, is a basidiomycetous oleaginous yeast known to accumulate lipids to more than 70% of its dry cell weight. The oleaginous red yeast (R. toruloides) has not been intensely studied in the lifespan domain. We designed this work to investigate how GG and proline promote the longevity of this red yeast strain. The results obtained in our study confirmed that these molecules increased R. toruloides' viability, survival percentage, and lifespan upon supplementation. GG exerts the most promising effects at a relatively high concentration (100 mM), while proline functions best at a low level (2 mM). Elucidation of the processes underlying these favorable responses revealed that GG promotes the yeast chronological lifespan (CLS) through increased catalase activity, modulation of the culture medium pH, a rise in ATP, and an increase in reactive oxygen species (ROS) accumulation (mitohormesis). It is critical to understand the mechanisms of these geroprotector molecules, particularly GG, and the proclivity of its lifespan application; this will aid in offering clarity on its potential application in aging research.

Modularly engineering Rhodotorula toruloides for α-terpineol production.

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5 mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds.

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide.

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola.

The sophorolipid-producing strain Starmerella bombicola CGMCC 1576 has a remarkable ability to produce sophorolipids (SLs) under the acidic and lactonic forms with almost equal proportion. In this study, we found the gene encoding for the long-chain acyl-CoA synthetase (ALCS). This enzyme was putatively identified as a membrane-bound long-chain fatty acid transport protein and contributed to the uptake of long-chain fatty acids. Disruption of the alcs gene resulted in an impaired growth of the alcs-deleted mutant in minimal media containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the fluorescent-tagged long-chain fatty acid analogue-boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823). The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of the SLs produced by the gene-deleted mutant of ∆alcs::six showed almost no lactonic form of SLs, and the acidic SLs were composed of medium-chain. The ALCS enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with pMAL-c2x-alcs. The enzyme was purified through a maltose-binding protein (MBP) affinity chromatography column and was confirmed to be homogeneous by SDS-PAGE. The recombinant enzyme could catalyze the formation of the long-chain acyl-CoA when the long-chain fatty acids and the coenzyme A were used as substrates.

Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum.

Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose.