Engineering CREB-activated promoters for adenosine-induced gene expression.

Accumulation of the ribonucleoside, adenosine (ADO), triggers a cAMP response element binding protein (CREB)-mediated signaling pathway to suppress the function of immune cells in tumors. Here, we describe a collection of CREB-activated promoters that allow for strong and tunable ADO-induced gene expression in human cells. By optimizing number of CREB transcription factor binding sites and altering the core promoter region of CREB-based hybrid promoters, we created synthetic constructs that drive gene expression to higher levels than strong, endogenous mammalian promoters in the presence of ADO. These synthetic promoters are induced up to 47-fold by ADO, with minimal expression in their "off" state. We further determine that our CREB-based promoters are activated by other compounds that act as signaling analogs, and that combinatorial addition of ADO and these compounds has a synergistic impact on gene expression. Surprisingly, we also detail how background ADO degradation caused by the common cell culture media additive, fetal bovine serum (FBS), confounds experiments designed to determine ADO dose-responsiveness. We show that only after long-term heat deactivation of FBS can our synthetic promoters enable gene expression induction at physiologically relevant levels of ADO. Finally, we demonstrate that the strength of a CREB-based promoter is enhanced by incorporating other transcription factor binding sites.

Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant.

Homo sapiens adenosine deaminase isoform 1 (HsADA1) hydrolyzes adenosine and 2-deoxyadenosine as a key step in the purine nucleoside salvage pathway. Some HsADA1 mutations have severe deleterious effects, as is the case in a severe combined immunodeficiency resulting from loss of enzyme activity (ADA-SCID). Other mutations that reduce enzyme activity, for instance the Asp8Asn (D8N) variant, do not cause ADA-SCID but are correlated with other consequences to health. To ease further study of HsADA1 and its variants, we optimized an inexpensive, recombinant expression process in an Escherichia coli host through multiplexed parameter testing enabled by a lysate-based microtiter plate assay. We demonstrate the importance of gene codon usage, induction time and temperature, and alcohol supplementation towards improving enzyme yield to a final titer of 5 mg per liter of culture. We further show that use of a double-histidine-tag (his-tag) system greatly improves purity. We then utilize our expression and purification framework to produce the HsADA1 D8N variant, which had previously not been purified to homogeneity. We confirm that the D8N variant is ∼30% less active than the wildtype HsADA1 and show that it better retains its activity in human serum. Additionally, we show that both HsADA1 and the D8N variant have heightened activity in serum, driven in part by a previously undescribed phenomenon involving albumin. Therefore, this work presents a valuable process to produce HsADA1 that allows for insights into it and its variants' behavior. We also confirm the utility of lysate-based activity assays towards finding optimal E. coli expression conditions for enzymes and show how fusing his-tags in tandem can enhance product purity.

A Rapid Antibody Enhancement Platform in Saccharomyces cerevisiae Using an Improved, Diversifying CRISPR Base Editor.

The yeast Saccharomyces cerevisiae is commonly used to interrogate and screen protein variants and to perform directed evolution studies to develop proteins with enhanced features. While several techniques have been described that help enable the use of yeast for directed evolution, there remains a need to increase their speed and ease of use. Here we present yDBE, a yeast diversifying base editor that functions in vivo and employs a CRISPR-dCas9-directed cytidine deaminase base editor to diversify DNA in a targeted, rapid, and high-breadth manner. To develop yDBE, we enhanced the mutation rate of an initial base editor by employing improved deaminase variants and characterizing several scaffolded guide constructs. We then demonstrate the ability of the yDBE platform to improve the affinity of a displayed antibody scFv, rapidly generating diversified libraries and isolating improved binders via cell sorting. By performing high-throughput sequencing analysis of the high-activity yDBE, we show that it enables a mutation rate of 2.13 × 10-4 substitutions/bp/generation over a window of 100 bp. As yDBE functions entirely in vivo and can be easily programmed to diversify nearly any such window of DNA, we posit that it can be a powerful tool for facilitating a variety of directed evolution experiments.

High throughput mutagenesis and screening for yeast engineering.

The eukaryotic yeast Saccharomyces cerevisiae is a model host utilized for whole cell biocatalytic conversions, protein evolution, and scientific inquiries into the pathogenesis of human disease. Over the past decade, the scale and pace of such studies has drastically increased alongside the advent of novel tools for both genome-wide studies and targeted genetic mutagenesis. In this review, we will detail past and present (e.g., CRISPR/Cas) genome-scale screening platforms, typically employed in the context of growth-based selections for improved whole cell phenotype or for mechanistic interrogations. We will further highlight recent advances that enable the rapid and often continuous evolution of biomolecules with improved function. Additionally, we will detail the corresponding advances in high throughput selection and screening strategies that are essential for assessing or isolating cellular and protein improvements. Finally, we will describe how future developments can continue to advance yeast high throughput engineering.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

Catalytically active holo Homo sapiens adenosine deaminase I adopts a closed conformation.

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Protein engineering: a driving force toward synthetic immunology.

The full application of the diverse toolkit of protein engineering has made it easier to control the immune system. In particular, synthetic cytokine variants and engineered immune receptor platforms have shown promise for the treatment of various indications with dysregulated immune function, particularly cancer. Here, we review recent advances in the control of immune cell signaling and therapeutic potency that have employed protein engineering strategies. We further discuss how safety concerns are driving the design of immunotherapeutics toward 'user-defined' control or requiring multiple distinct inputs before a functional response, highlighting emergent control strategies employed for chimeric antigen receptor (CAR) engineering.

Immunosuppressive metabolites in tumoral immune evasion: redundancies, clinical efforts, and pathways forward.

Tumors accumulate metabolites that deactivate infiltrating immune cells and polarize them toward anti-inflammatory phenotypes. We provide a comprehensive review of the complex networks orchestrated by several of the most potent immunosuppressive metabolites, highlighting the impact of adenosine, kynurenines, prostaglandin E2, and norepinephrine and epinephrine, while discussing completed and ongoing clinical efforts to curtail their impact. Retrospective analyses of clinical data have elucidated that their activity is negatively associated with prognosis in diverse cancer indications, though there is a current paucity of approved therapies that disrupt their synthesis or downstream signaling axes. We hypothesize that prior lukewarm results may be attributed to redundancies in each metabolites' synthesis or signaling pathway and highlight routes for how therapeutic development and patient stratification might proceed in the future.

Advances in promoter engineering: Novel applications and predefined transcriptional control.

Synthetic biology continues to progress by relying on more robust tools for transcriptional control, of which promoters are the most fundamental component. Numerous studies have sought to characterize promoter function, determine principles to guide their engineering, and create promoters with stronger expression or tailored inducible control. In this review, we will summarize promoter architecture and highlight recent advances in the field, focusing on the novel applications of inducible promoter design and engineering towards metabolic engineering and cellular therapeutic development. Additionally, we will highlight how the expansion of new, machine learning techniques for modeling and engineering promoter sequences are enabling more accurate prediction of promoter characteristics.

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Metabolic engineering of Yarrowia lipolytica for itaconic acid production.

Itaconic acid is a naturally produced organic acid with diverse applications as a replacement for petroleum derived products. However, its industrial viability as a bio-replacement has been restricted due to limitations with native producers. In this light, Yarrowia lipolytica is an excellent potential candidate for itaconic acid production due to its innate capacity to accumulate citric acid cycle intermediates and tolerance to lower pH. Here, we demonstrate the capacity to produce itaconic acid in Y. lipolytica through heterologous expression of the itaconic acid synthesis enzyme, resulting in an initial titer of 33 mg/L. Further optimizations of this strain via metabolic pathway engineering, enzyme localization, and media optimization strategies enabled 4.6g/L of itaconic acid to be produced in bioreactors, representing a 140-fold improvement over initial titer. Moreover, these fermentation conditions did not require additional nutrient supplementation and utilized a low pH condition that enabled the acid form of itaconic acid to be produced. Overall yields (0.058 g/g yield from glucose) and maximum productivity of 0.045 g/L/h still provide areas for future strain improvement. Nevertheless, this work demonstrates that Y. lipolytica has the potential to serve as an industrially relevant platform for itaconic acid production.

Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant.

Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor. The resulting strain is enriched in unsaturated fatty acids. Comparing the underlying mechanism of this mutant to other previously engineered strains suggests that creating an imbalance between glycolysis and the TCA cycle can serve as a driving force for lipogenesis when combined with fatty acid catabolism overexpressions. Further comparative transcriptomics analysis revealed both distinct and convergent rewiring associated with these different genotypes. Finally, by combining metabolic engineering targets, it is possible to further engineer a strain containing the mutant mga2 gene to a lipid production titer of 25g/L.

Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production.

Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. Here, we utilize Saccharomyces cerevisiae to enable the synthesis of itaconic acid, a molecule with diverse applications as a petrochemical replacement. We first optimize pathway expression within S. cerevisiae through the use of a hybrid promoter. Next, we utilize sequential, in silico computational genome-scanning to identify beneficial genetic perturbations that are metabolically distant from the itaconic acid synthesis pathway. In this manner, we successfully identify three non-obvious genetic targets (∆ade3 ∆bna2 ∆tes1) that successively improve itaconic acid titer. We establish that focused manipulations of upstream pathway enzymes (localized refactoring) and enzyme re-localization to both mitochondria and cytosol fail to improve itaconic acid titers. Finally, we establish a higher cell density fermentation that ultimately achieves itaconic acid titer of 168 mg/L, a sevenfold improvement over initial conditions. This work represents an attempt to increase itaconic acid production in yeast and demonstrates the successful utilization of computationally guided genetic manipulation to increase metabolic capacity.

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production.

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l(-1) lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.

Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica.

The complete biosynthetic replacement of petroleum transportation fuels requires a metabolic pathway capable of producing short chain n-alkanes. Here, we report and characterize a proof-of-concept pathway that enables microbial production of the C5 n-alkane, pentane. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid byproduct. Initial expression of the soybean lipoxygenase enzyme within a Yarrowia lipolytica host yielded 1.56 mg/L pentane. Efforts to improve pentane yield by increasing substrate availability and strongly overexpressing the lipoxygenase enzyme successfully increased pentane production three-fold to 4.98 mg/L. This work represents the first-ever microbial production of pentane and demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. In this regard, we demonstrate the potential pliability of Y. lipolytica toward the biosynthetic production of value-added molecules from its generous fatty acid reserves.

Promoter engineering: recent advances in controlling transcription at the most fundamental level.

Synthetic control of gene expression is critical for metabolic engineering efforts. Specifically, precise control of key pathway enzymes (heterologous or native) can help maximize product formation. The fundamental level of transcriptional control takes place at promoter elements that drive gene expression. Endogenous promoters are limited in that they do not fully sample the complete continuum of transcriptional control, and do not maximize the transcription levels achievable within an organism. To address this issue, several attempts at promoter engineering have shown great promise both in expanding the cell-wide transcriptional capacity of an organism and in enabling tunable levels of gene expression. Thus, this review highlights the recent advances and approaches for altering gene expression control at the promoter level. Furthermore, we propose that recent advances in the understanding of transcription factors and their DNA-binding sites will enable rational and predictive control of gene expression.

Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica.

Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters-the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UASTEF#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UASTEF#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UASTEF and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.

Linking yeast Gcn5p catalytic function and gene regulation using a quantitative, graded dominant mutant approach.

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity--all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence.

Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters.

A dynamic range of well-controlled constitutive and tunable promoters are essential for metabolic engineering and synthetic biology applications in all host organisms. Here, we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two modular components-the enhancer element, consisting of tandem repeats or combinations of upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility of this approach with three main case studies. First, we establish a dynamic range of constitutive promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to impart synthetic regulation through a hybrid promoter approach by adding galactose activation and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid promoter approach can expand, enhance, and control promoter activity.