Medium composition overturns the widely accepted sulfate-dependent repression of desulfurization phenotype in Rhodococcus qingshengii IGTS8.

Microbial desulfurization has been extensively studied as a promising alternative to the widely applied chemical desulfurization process. Sulfur removal from petroleum and its products becomes essential, as the environmental regulations become increasingly stringent. Rhodococcus qingshengii IGTS8 has gained ground as a naturally occurring model biocatalyst, due to its superior specific activity for desulfurization of dibenzothiophene (DBT). Recalcitrant organic sulfur compounds-DBT included-are preferentially removed by selective carbon-sulfur bond cleavage to avoid a reduction in the calorific value of the fuel. The process, however, still has not reached economically sustainable levels, as certain limitations have been identified. One of those bottlenecks is the repression of catalytic activity caused by ubiquitous sulfur sources such as inorganic sulfate, methionine, or cysteine. Herein, we report an optimized culture medium for wild-type stain IGTS8 that completely alleviates the sulfate-mediated repression of biodesulfurization activity without modification of the natural biocatalyst. Medium C not only promotes growth in the presence of several sulfur sources, including DBT, but also enhances biodesulfurization of resting cells grown in the presence of up to 5 mM sulfate. Based on the above, the present work can be considered as a step towards the development of a more viable commercial biodesulfurization process.

Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach.

Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter Pdsz, with the nonrepressible Pkap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

Interplay between Sulfur Assimilation and Biodesulfurization Activity in Rhodococcus qingshengii IGTS8: Insights into a Regulatory Role of the Reverse Transsulfuration Pathway.

Biodesulfurization is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts harboring the highly conserved desulfurization operon dszABC, which is significantly repressed by methionine, cysteine, and inorganic sulfate, have been isolated. However, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the reverse transsulfuration pathway enzyme genes cbs and metB were constructed in the desulfurizing strain Rhodococcus sp. strain IGTS8. We provide sequence analyses and report the enzymes' involvement in the sulfate- and methionine-dependent repression of biodesulfurization activity. Sulfate addition in the bacterial culture did not repress the desulfurization activity of the Δcbs strain, whereas deletion of metB promoted a significant biodesulfurization activity for sulfate-based growth and an even higher desulfurization activity for methionine-grown cells. In contrast, growth on cysteine completely repressed the desulfurization activity of all strains. Transcript level comparison uncovered a positive effect of cbs and metB gene deletions on dsz gene expression in the presence of sulfate and methionine, but not cysteine, offering insights into a critical role of cystathionine ß-synthase (CßS) and MetB in desulfurization activity regulation. IMPORTANCE Precise genome editing of the model biocatalyst Rhodococcus qingshengii IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of dsz gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, CßS and MetB. Moreover, we observed an enhancement of biodesulfurization capability in the presence of otherwise repressive sulfur sources, such as sulfate and l-methionine. The interconnection of cellular sulfur assimilation strategies was revealed and validated.

Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment.

Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.

Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

Nutrient transporters, being polytopic membrane proteins, are believed, but not formally shown, to traffic from their site of synthesis, the ER, to the plasma membrane through Golgi-dependent vesicular trafficking. Here, we develop a novel genetic system to investigate the trafficking of a neosynthesized model transporter, the well-studied UapA purine transporter of Aspergillus nidulans. We show that sorting of neosynthesized UapA to the plasma membrane (PM) bypasses the Golgi and does not necessitate key Rab GTPases, AP adaptors, microtubules or endosomes. UapA PM localization is found to be dependent on functional COPII vesicles, actin polymerization, clathrin heavy chain and the PM t-SNARE SsoA. Actin polymerization proved to primarily affect COPII vesicle formation, whereas the essential role of ClaH seems indirect and less clear. We provide evidence that other evolutionary and functionally distinct transporters of A. nidulans also follow the herein identified Golgi-independent trafficking route of UapA. Importantly, our findings suggest that specific membrane cargoes drive the formation of distinct COPII subpopulations that bypass the Golgi to be sorted non-polarly to the PM, and thus serving house-keeping cell functions.

Transporter membrane traffic and function: lessons from a mould.

Transporters are essential transmembrane proteins that mediate the selective translocation of solutes, ions or drugs across biological membranes. Their function is related to cell nutrition, communication, stress resistance and homeostasis. Consequently, their malfunction is associated with genetic or metabolic diseases and drug sensitivity or resistance. A distinctive characteristic of transporters is their cotranslational translocation and folding in a membrane bilayer, this being the endoplasmic reticulum (ER) in eukaryotes or the cell membrane in prokaryotes. In the former case, transporters exit the ER packed in secretory vesicles and traffic via seemingly unconventional, rather than Golgi-dependent, sorting routes to their final destination, the plasma membrane (PM). Proper folding is a prerequisite for ER exit and further trafficking. Misfolded transporters, either due to mutations, high temperature of chemical agents (e.g. DMSO, DTT) are blocked in the ER. The accumulation of ER-retained transporters, in most cases, elicits endoplasmic reticulum-associated degradation, but also ubiquitination-dependent, chaperone-mediated, selective autophagy. The function of PM transporters is finely regulated at the cellular level, in response to physiological or stress signals that promote, via α-arrestin-assisted ubiquitination, their endocytosis and vacuolar/lysosomal degradation, and in some cases recycling to the PM. Importantly, transporter oligomerization and specific interactions with membrane lipids are emerging as important players in transporter expression, function and turnover. This review discusses how paradigmatic work on transporters of a model mould, Aspergillus nidulans, has contributed to novel findings related to transporter functioning in eukaryotes.

Secretory Vesicle Polar Sorting, Endosome Recycling and Cytoskeleton Organization Require the AP-1 Complex in Aspergillus nidulans.

The AP-1 complex is essential for membrane protein traffic via its role in the pinching-off and sorting of secretory vesicles (SVs) from the trans-Golgi and/or endosomes. While its essentiality is undisputed in metazoa, its role in simpler eukaryotes seems less clear. Here, we dissect the role of AP-1 in the filamentous fungus Aspergillus nidulans and show that it is absolutely essential for growth due to its role in clathrin-dependent maintenance of polar traffic of specific membrane cargoes toward the apex of growing hyphae. We provide evidence that AP-1 is involved in both anterograde sorting of RabERab11-labeled SVs and RabA/BRab5-dependent endosome recycling. Additionally, AP-1 is shown to be critical for microtubule and septin organization, further rationalizing its essentiality in cells that face the challenge of cytoskeleton-dependent polarized cargo traffic. This work also opens a novel issue on how nonpolar cargoes, such as transporters, are sorted to the eukaryotic plasma membrane.

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi.

Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 ß subunit (ß2) of higher fungi lacks a clathrin-binding domain, and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaBEnd4 and SagAEnd3 and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition is further supported by its functional interaction with BasA (sphingolipid biosynthesis) and StoA (apical sterol-rich membrane domains), and its essentiality in polar deposition of chitin. Our findings support that the AP-2 complex of dikarya has acquired, in the course of evolution, a specialized clathrin-independent function necessary for fungal polar growth.

BsdA(Bsd2) -dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy.

Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non-endocytic vacuolar turnover of ΔR481 is dependent on BsdA(Bsd2) , an ER transmembrane adaptor of HulA(Rsp5) ubiquitin ligase. We obtain in vivo evidence that BsdA(Bsd2) interacts with HulA(Rsp5) and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO(ts) ) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.

Oligomerization of the UapA Purine Transporter Is Critical for ER-Exit, Plasma Membrane Localization and Turnover.

Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.