James M. Wagner

Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances

Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future.

Biosensor-Enabled Directed Evolution to Improve Muconic Acid Production in Saccharomyces cerevisiae

Muconic acid is a valuable platform chemical with potential applications in the production of polymers such as nylon and polyethylene terephthalate (PET). The conjugate base, muconate, has been previously biosynthesized in the bacterial host Escherichia coli. Likewise, previous significant pathway engineering lead to the first reported instance of rationally engineered production of muconic acid in the yeast Saccharomyces cerevisiae. To further increase muconic acid production in this host, a combined adaptive laboratory evolution (ALE) strategy and rational metabolic engineering is employed. To this end, a biosensor module that responds to the endogenous aromatic amino acid (AAA) as a surrogate for pathway flux is adapted. Following two rounds of ALE coupled with an anti‐metabolite feeding strategy, the strains with improved AAA pathway flux is isolated. Next, it is demonstrated that this increased flux can be redirected into the composite muconic acid pathway with a threefold increase in the total titer of the composite pathway compared to our previously engineered strain. Finally, a truncation of the penta‐functional ARO1 protein is complemented and overexpress an endogenous aromatic decarboxylase to establish a final strain capable of producing 0.5 g L−1 muconic acid in shake flasks and 2.1 g L−1 in a fed‐batch bioreactor with a yield of 12.9 mg muconic acid/g glucose at the rate of 9.0 mg h−1. This value represents the highest titer of muconic acid reported to date in S. cerevisiae, in addition to the highest reported titer of a shikimate pathway derivative in this host.

In vivo continuous evolution of genes and pathways in yeast

Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107 l−1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo.

Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications

Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection. We focus on the use of these methods for strain engineering and enzyme discovery and conclude with remarks on the current and future state of biosensor development for application in the metabolic engineering field.

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes

Synthetic biology and metabolic engineering seek to re-engineer microbes into “living foundries” for the production of high value chemicals. Through a “design-build-test” cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

Significance Polyketides are important molecules for both their bioactive traits and their potential as chemical building blocks. However, production of these molecules through chemistry and biocatalysts is restricted in yield and titer. Here, we demonstrate that the nonconventional yeast Yarrowia lipolytica can serve as a potent host for such production. This work provides a comprehensive evaluation of three separate pathways toward acetyl–CoA and malonyl–CoA in this host, enabling high-titer production of triacetic acid lactone. Beyond achieving unprecedented titers and appreciable yields, this production capacity allows for both purification from fermentation broth and conversion into a material using simple reaction conditions. Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl–CoA. By exploring three distinct metabolic engineering strategies for acetyl–CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.

Developing a piggyBac Transposon System and Compatible Selection Markers for Insertional Mutagenesis and Genome Engineering in Yarrowia lipolytica

Yarrowia lipolytica is a non-conventional yeast of interest to the biotechnology industry. However, the physiology, metabolism, and genetic regulation of Y. lipolytica diverge significantly from more well-studied and characterized yeasts such as Saccharomyces cerevisiae. To develop additional genetic tools for this industrially relevant host, the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and introduction of scarless, footprint-free genomic modifications in Y. lipolytica. Specifically, we demonstrate piggyBac transposition in Y. lipolytica, and then use the approach to screen transposon insertion libraries for rapid isolation of mutations that confer altered canavanine resistance, pigment formation, and neutral lipid accumulation. We also develop a variety of piggyBac compatible selection markers for footprint-free genome engineering, including a novel dominant marker cassette (Escherichia coli guaB) for effective Y. lipolytica selection using mycophenolic acid. We utilize these marker cassettes to construct a piggyBac vector set that allows for auxotrophic selection (uracil or tryptophan biosynthesis) or dominant selection (hygromycin, nourseothricin, chlorimuron ethyl, or mycophenolic acid resistance) and subsequent marker excision. These new genetic tools and techniques will help to facilitate and accelerate the engineering of Y. lipolytica strains for efficient and sustainable production of a wide variety of small molecules and proteins.

A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening.

Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. To address this limitation, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which functional determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified mutations that led to high lipid accumulation and eliminated pseudohyphal morphology.