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The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(DL-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Čerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.

The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica is developed and is used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators are compared for their effectiveness in Y. lipolytica, with the synthetic tripartite activator VPR yielding the highest activation. A VPR-dCas9 fusion is then targeted to various locations in a synthetic promoter driving hrGFP expression, and activation is achieved. Subsequently, the CRISPRa system is used to activate transcription of two different native β-glucosidase genes, facilitating enhanced growth on cellobiose as the sole carbon source. This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.

Background The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis. Results Enabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR–Cas9 system from Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde. Conclusions Newly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPR–Cas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1–7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0854-5) contains supplementary material, which is available to authorized users.

In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4+/− B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4F) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4F dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation.

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

Gonadectomy (GDX) induces sex steroid-producing adrenocortical tumors in certain mouse strains and in the domestic ferret. Transcriptome analysis and DNA methylation mapping were used to identify novel genetic and epigenetic markers of GDX-induced adrenocortical neoplasia in female DBA/2J mice. Markers were validated using a combination of laser capture microdissection, quantitative RT-PCR, in situ hybridization, and immunohistochemistry. Microarray expression profiling of whole adrenal mRNA from ovariectomized vs. intact mice demonstrated selective upregulation of gonadal-like genes including Spinlw1 and Insl3 in GDX-induced adrenocortical tumors of the mouse. A complementary candidate gene approach identified Foxl2 as another gonadal-like marker expressed in GDX-induced neoplasms of the mouse and ferret. That both “male-specific” (Spinlw1) and “female-specific” (Foxl2) markers were identified is noteworthy and implies that the neoplasms exhibit mixed characteristics of male and female gonadal somatic cells. Genome-wide methylation analysis showed that two genes with hypomethylated promoters, Igfbp6 and Foxs1, are upregulated in GDX-induced adrenocortical neoplasms. These new genetic and epigenetic markers may prove useful for studies of steroidogenic cell development and for diagnostic testing.

DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine–guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.