Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Citation: Tran, T. N. T., Breuer, R. J., Narasimhan, R. A., Parreiras, L. S., Zhang, Y. P., Sato, T. K., & Durrett, T. P. (2017). Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose. Biotechnology for Biofuels, 10, 12. doi:10.1186/s13068-017-0751-yBackground: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. Results: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media. Conclusion: Our results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids

Targeted Imaging of Urothelium Carcinoma in Human Bladders by an ICG pHLIP Peptide Ex vivo

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH Low Insertion Peptide (pHLIP® peptide) conjugated with a near infrared fluorescent dye (ICG) targets low extracellular pH allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with non-buffered saline and instilled with a solution of the ICG pHLIP® construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP® imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80%, if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. ICG pHLIP® imaging agent marked high grade urothelial carcinomas, both muscle invasive and non-muscle invasive. Carcinoma in situ (CIS) was accurately diagnosed in 11 cases, whereas only 4 cases were seen using white light, so imaging with the ICG pHLIP® peptide offers improved early diagnosis of bladder cancers, and may also enable new treatment alternatives

Phenolic Impregnated Carbon Ablators (PICA) as Thermal Protection Systems for Discovery Missions

This paper presents the development of the light weight Phenolic Impregnated Carbon Ablators (PICA) and its thermal performance in a simulated heating environment for planetary entry vehicles. The PICA material was developed as a member of the Light Weight Ceramic Ablators (LCA's), and the manufacturing process of this material has since been significantly improved. The density of PICA material ranges from 14 to 20 lbm/ft(exp 3), having uniform resin distribution with and without a densified top surface. The thermal performance of PICA was evaluated in the Ames arc-jet facility at cold wall heat fluxes from 375 to 2,960 BtU/ft(exp 2)-s and surface pressures of 0.1 to 0.43 atm. Heat loads used in these tests varied from 5,500 to 29,600 BtU/ft(exp 2) and are representative of the entry conditions of the proposed Discovery Class Missions. Surface and in-depth temperatures were measured using optical pyrometers and thermocouples. Surface recession was also measured by using a template and a height gage. The ablation characteristics and efficiency of PICA are quantified by using the effective heat of ablation, and the thermal penetration response is evaluated from the thermal soak data. In addition, a comparison of thermal performance of standard and surface densified PICA is also discussed

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation

The surface receptor CD8α is present on 20%-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α- (persistent CD8α-). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell-activating receptors

Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy

Anaplastic Transformation in Thyroid Cancer Revealed by Single-Cell Transcriptomics

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intratumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single-cell transcriptomic and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), and mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Furthermore, our analysis identified 2 important milestones: (a) a diploid stage, in which iATC cells were diploids with inflammatory phenotypes and (b) an aneuploid stage, in which mATCs gained aneuploid genomes and mesenchymal phenotypes, producing excessive amounts of collagen and collagen-interacting receptors. In parallel, cancer-associated fibroblasts showed strong interactions among mesenchymal cell types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for the treatment of ATC

Memory-like differentiation enhances NK cell responses to melanoma

PURPOSE: Treatment of advanced melanoma is a clinical challenge. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently deficient or dysfunctional in patients with melanoma. Thus, new strategies are needed to enhance NK-cell antitumor responses. Cytokine-induced memory-like (ML) differentiation overcomes many barriers in the NK-cell therapeutics field, resulting in potent cytotoxicity and enhanced cytokine production against blood cancer targets. However, the preclinical activity of ML NK against solid tumors remains largely undefined. EXPERIMENTAL DESIGN: Phenotypic and functional alterations of blood and advanced melanoma infiltrating NK cells were evaluated using mass cytometry. ML NK cells from healthy donors (HD) and patients with advanced melanoma were evaluated for their ability to produce IFNγ and kill melanoma targets RESULTS: NK cells in advanced melanoma exhibited a decreased cytotoxic potential compared with blood NK cells. ML NK cells differentiated from HD and patients with advanced melanoma displayed enhanced IFNγ production and cytotoxicity against melanoma targets. This included ML differentiation enhancing melanoma patients\u27 NK-cell responses against autologous targets. The ML NK-cell response against melanoma was partially dependent on the NKG2D- and NKp46-activating receptors. Furthermore, in xenograft NSG mouse models, human ML NK cells demonstrated superior control of melanoma, compared with conventional NK cells. CONCLUSIONS: Blood NK cells from allogeneic HD or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials

Memory-like differentiation enhances NK cell responses against colorectal cancer

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB

T-BET and EOMES sustain mature human NK cell identity and antitumor function

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity

Androgen receptor signaling regulates the transcriptome of prostate cancer cells by modulating global alternative splicing

Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX®) and enzalutamide (XTANDI®). However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8–19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide