A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

All authors are with the Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA -- Hal S. Alper is with the Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA -- Amanda M. Lanza Current Address: Bristol-Myers Squibb, Biologics Development, 35 South Street, Hopkinton, MA 01748, USABackground: Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results: Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions: Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.Chemical EngineeringInstitute for Cellular and Molecular [email protected]

Engineered xylose transporters with reduced glucose inhibition

Provided herein are compositions and methods useful for reducing glucose inhibition in transporting xylose, arabinose and other monosaccharides, into a yeast cell.Board of Regents, University of Texas Syste

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

Exogenous terminators for controlling fungal gene expression

Provided herein are exogenous terminator sequences for use in fungi cell transcriptional termination.Board of Regents, University of Texas Syste

Expanding beyond canonical metabolism: Interfacing alternative elements, synthetic biology, and metabolic engineering

Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner. However, most industrial applications have focused on only a small subset of elements from the periodic table, centered around carbon biochemistry. This review aims to illustrate the expanse of chemical elements that can currently (and potentially) be integrated into useful products using cellular systems. Specifically, we describe recent advances in expanding the cellular scope to include the halogens, selenium and the metalloids, and a variety of metal incorporations. These examples range from small molecules, heteroatom-linked uncommon elements, and natural products to biomining and nanotechnology applications. Collectively, this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology

Metabolic engineering of microbial cell factories for production of nutraceuticals

Abstract Metabolic engineering allows for the rewiring of basic metabolism to overproduce both native and non-native metabolites. Among these biomolecules, nutraceuticals have received considerable interest due to their health-promoting or disease-preventing properties. Likewise, microbial engineering efforts to produce these value-added nutraceuticals overcome traditional limitations of low yield from extractions and complex chemical syntheses. This review covers current strategies of metabolic engineering employed for the production of a few key nutraceuticals with selecting polyunsaturated fatty acids, polyphenolic compounds, carotenoids and non-proteinogenic amino acids as exemplary molecules. We focus on the use of both mono-culture and co-culture strategies to produce these molecules of interest. In each of these cases, metabolic engineering efforts are enabling rapid production of these molecules

Directed Evolution Of Xylose Isomerase For Improved Xylose Catabolism And Fermentation In The Yeast Saccharomyces Cerevisiae

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tall and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.University of Texas at Austin Research Grant ProgramCivil, Architectural, and Environmental EngineeringChemical Engineerin

Transcriptomics-Guided Design of Synthetic Promoters for a Mammalian System

Despite recent advances in improving titers for therapeutic proteins such as antibodies to the 10 g/L scale, these high yields can only be achieved in select mammalian hosts. Regardless of the host or product, strong promoters are required to obtain high levels of transgene expression. However, the promoters employed to drive this expression are rather limited in variety and are usually either viral-derived or screened empirically during vector design. To begin to move away from viral parts, we employed a more systematic approach to identify and design new synthetic promoters using endogenous elements. To do so, we established a workflow to design these elements by (1) analyzing the transcriptomics profile of a specific cell line under a desired, representative cell culture condition, (2) identifying key genetic motifs using bioinformatics that can be used to rationally construct synthetic promoters, (3) building synthetic promoters using conventional DNA synthesis and molecular biology techniques, and (4) evaluating the performance of these synthetic promoters using model proteins. The resulting promoters perform comparably to the hCMV IE promoter variants tested, but with endogenous components. During this design–build–test cycle we also investigated the underlying design rules for transcription factor binding site arrangement in synthetic promoters. Overall, this approach of using an “omics-guided” workflow for designing synthetic promoters facilitates the construction of high expression vectors for immediate use in current production hosts