Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Shifting the distribution: modulation of the lipid profile in Yarrowia lipolytica via iron content.

Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: • Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. • Iron addition reversibly alters lipid portfolio increasing linolenic acid. • Removal of iron from YPD media provides a link to enhanced oleochemical production.

Metabolic engineering of microbial cell factories for production of nutraceuticals.

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.

Expanding the Chemical Palette of Industrial Microbes: Metabolic Engineering for Type III PKS-Derived Polyketides.

Polyketides are a unique class of molecules with attractive bioactive and chemical properties. As a result, biorenewable production is being explored with these molecules as potential pharmaceutical, fuel, and material precursors. In particular, type III polyketide synthases enable access to a diverse class of chemicals using a relatively simple biochemical synthesis pathway. In this review, the recent advances in the engineering of microbial hosts for the production of type III PKS-derived polyketides are highlighted. In particular, the field has moved beyond simple proof-of-concept and has been exploring engineering efforts that have led to improved production scales. This review details engineering progress for the production of acetyl-CoA- and malonyl-CoA-derived polyketides including the products triacetic acid lactone and phloroglucinol as well as polyphenolic, phenylpropanoid-derived compounds including flavonoids, stilbenoids, and curcuminoids. Specifically, the authors focus on enumerating the metabolic engineering strategies employed and product titers achieved for these molecules. Finally, the authors highlight tools and strategies that can be leveraged to realize the potential of microbial production and diversification of these molecules.

Engineering a Glucosamine-6-phosphate Responsive glmS Ribozyme Switch Enables Dynamic Control of Metabolic Flux in Bacillus subtilis for Overproduction of N-Acetylglucosamine.

Bacillus subtilis is a typical industrial microorganism and is widely used in industrial biotechnology, particularly for nutraceutical production. There are many studies on the static metabolic engineering of B. subtilis, whereas there are few reports on dynamic metabolic engineering due to the lack of appropriate elements. Here, we established a dynamic reprogramming strategy for reconstructing metabolic networks in B. subtilis, using a typical nutraceutical, N-acetylglucosamine (GlcNAc), as a model product and the glmS (encoding glucosamine-6-phosphate synthase) ribozyme as an engineering element. First, a trp terminator was introduced to effectively release the glmS ribozyme feedback inhibition. Further, we engineered the native glucosamine-6-phosphate (GlcN6P) responsive glmS ribozyme switch to dynamically control the metabolic flux in B. subtilis for overproduction of GlcNAc. With GlcN6P as a ligand, the native sensor glmS ribozyme is integrated at the 5'- of phosphoglucosamine mutase and 6-phosphofructokinase genes to decrease the flux dynamically toward the peptidoglycan synthesis and glycolysis pathway, respectively. The glmS ribozyme mutant M5 ( glmS ribozyme cleavage site AG → GG) with decreased ribozyme activity is integrated at the 5'- of glucose-6-phosphate isomerase gene to increase the flux dynamically toward the GlcNAc synthesis pathway. This strategy increased the GlcNAc titer from 9.24 to 18.45 g/L, and the specific GlcNAc productivity from 0.53 to 1.21 g GlcNAc/g cell. Since GlcN6P is involved in the biosynthesis of various products, here the developed strategy for multiple target dynamic engineering of metabolic pathways can be generally used in B. subtilis and other industrial microbes for chemical production.

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.

Using the Cre/lox system for targeted integration into the human genome: loxFAS-loxP pairing and delayed introduction of Cre DNA improve gene swapping efficiency.

Cre recombinase is a commonly-used genome editing tool suitable for site-specific integrations in mammalian genomes; however, the efficiency of transgenic swapping events compared to excision remains limited. Here we sought to identify important parameters and limiting factors that influence swapping propensity in this system, especially when using one wild-type loxP site. To modulate and increase the occurrence of swapping events, we identified two novel parameters. First, we identified the loxFAS-loxP pairing, a sequence never before used in mammalian systems, as the best choice for increasing swapping events in human cell lines. Second, for the first time we implicate the importance of delayed introduction of Cre DNA for optimal swapping efficiency. This same modification could potentially be of use to other systems catalyzing trimolecular reactions such as ΦC31 integrase and FLP recombinase where we hypothesize that transport of the exchange cassette is likewise initially rate limiting. The total number of recombination events, but not the ratio of swapping to excision, was found to be influenced by the quantity of Cre DNA transfected. Through this study, we were able to obtain Cre-mediated swapping frequencies of 8-12% without antibiotic enrichment, which represents nearly an order of magnitude increase over prior reports in the literature.