Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

The science of Durban, AIDS 2016.

INTRODUCTION: The science presented at the 21st International AIDS Conference in Durban, South Africa, in July 2016, addressed the state of the field across basic, clinical, prevention, law and policy and implementation science. METHODS AND RESULTS: The AIDS response has seen remarkable achievements in scientific advances, in translation of those advances into prevention, treatment and care for affected individuals and communities, and in large scale implementation - reaching 18 million people with antiviral therapy by mid-year 2016. Yet incident HIV infections in adults remain stubbornly stable and are increasing in some regions and among adolescents and adults in some key populations, challenging current science, policy and programming. There have been important advances in both preventive vaccines and in cure research, but both areas require ongoing investment and innovation. Clinical research has flourished with new agents, regimens, delivery modes and diagnostics but has been challenged by aging and increasingly complex patient populations, long-term adherence challenges, co-infections and co-morbidities, and unresolved issues in TB management and epidemic control. It is an extraordinary period of innovation in prevention, yet the promise of new tools and combination approaches have yet to deliver epidemic HIV control. CONCLUSIONS: Proven interventions, most notably pre-exposure prophylaxis, PrEP, have been limited in rollout and impact. Treatment as prevention has the promise to improve clinical outcomes but remains uncertain as a prevention tool to reduce population-level HIV incidence. The improvement of legal, policy and human rights environments for those most at risk for HIV acquisition and most at risk for lack of access to essential services; sexual and gender minorities, sex workers of all genders, people who inject drugs, and prisoners and detainees remain among the greatest unmet needs in HIV/AIDS. Failure to do better for these individuals and communities could undermine the HIV response.

The global response to HIV in men who have sex with men.

Gay, bisexual, and other men who have sex with men (MSM) continue to have disproportionately high burdens of HIV infection in countries of low, middle, and high income in 2016. 4 years after publication of a Lancet Series on MSM and HIV, progress on reducing HIV incidence, expanding sustained access to treatment, and realising human rights gains for MSM remains markedly uneven and fraught with challenges. Incidence densities in MSM are unacceptably high in countries as diverse as China, Kenya, Thailand, the UK, and the USA, with substantial disparities observed in specific communities of MSM including young and minority populations. Although some settings have achieved sufficient coverage of treatment, pre-exposure prophylaxis (PrEP), and human rights protections for sexual and gender minorities to change the trajectory of the HIV epidemic in MSM, these are exceptions. The roll-out of PrEP has been notably slow and coverage nowhere near what will be required for full use of this new preventive approach. Despite progress on issues such as marriage equality and decriminalisation of same-sex behaviour in some countries, there has been a marked increase in anti-gay legislation in many countries, including Nigeria, Russia, and The Gambia. The global epidemic of HIV in MSM is ongoing, and global efforts to address it remain insufficient. This must change if we are ever to truly achieve an AIDS-free generation.

CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.

Financing the response to HIV among gay men and other men who have sex with men: Case studies from eight diverse countries.

Despite reductions in the number of new HIV infections globally, the HIV epidemic among men who have sex with men (MSM) is expanding. This study characterises financing of HIV programmes for MSM and the impact of criminalisation on levels of funding, using data from five countries that criminalise same-sex sexual practices (Ethiopia, Mozambique, Guyana, India and Nigeria) and three that do not (China, Ukraine and Vietnam). For each country, all publicly available documents from the Global Fund to Fight AIDS, Tuberculosis and Malaria for approved HIV/AIDS grants in Rounds 5-9 and Country Operational Plans detailing investments made through the President's Emergency Plan for AIDS Relief (PEPFAR) from US fiscal year (FY) 2007-2009 were examined. Eleven of 20 HIV proposals to the Global Fund contained programmes for MSM totalling approximately $40 million or 6% of proposed budgets. In six countries providing activity-level data on MSM programming, PEPFAR funding that served this population and others ranged from $23.3 million in FY2007 to $35.4 million in FY2009, representing 0.5-25.9% of overall, non-treatment funding over this period. Countries that criminalise same-sex sexual practices spend fewer resources on HIV programmes serving MSM. However, they also show consistent underfunding of programmes serving MSM regardless of context or geography.

Multiplex engineering of industrial yeast genomes using CRISPRm.

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

Demand for programs for key populations in Africa from countries receiving international donor assistance.

There has been increasing attention in recent years to the HIV prevention, treatment, and care needs of key populations in Africa, in particular men who have sex with men (MSM), injection drug users (IDU), and female sex workers (FSW). While several major donors have undertaken efforts to prioritize these groups, it remains unclear which African countries are actively seeking donor support for these programs. For this analysis, we reviewed publicly available proposal and budget documentation from the US PEPFAR for fiscal years 2007 through 2010 and Rounds 1 through 10 of the Global Fund to Fight AIDS, Tuberculosis and Malaria for 40 countries in sub-Saharan Africa. Of the 164 searchable documents retrieved, nearly two-thirds contained at least one program serving FSW (65%, 107 proposals), less than one-third contained at least one program serving MSM (29%, 47 proposals), and a minority proposed programming for IDU (13%, 21 proposals). Demand for these programs was highly concentrated in a subset of countries. Epidemiological data for at least one key population was included in a majority of these proposals (63%, 67 proposals), but in many cases these data were not linked to programs.

Genetic variation in Saccharomyces cerevisiae: circuit diversification in a signal transduction network.

The connection between genotype and phenotype was assessed by determining the adhesion phenotype for the same mutation in two closely related yeast strains, S288c and Sigma, using two identical deletion libraries. Previous studies, all in Sigma, had shown that the adhesion phenotype was controlled by the filamentation mitogen-activated kinase (fMAPK) pathway, which activates a set of transcription factors required for the transcription of the structural gene FLO11. Unexpectedly, the fMAPK pathway is not required for FLO11 transcription in S288c despite the fact that the fMAPK genes are present and active in other pathways. Using transformation and a sensitized reporter, it was possible to isolate RPI1, one of the modifiers that permits the bypass of the fMAPK pathway in S288c. RPI1 encodes a transcription factor with allelic differences between the two strains: The RPI1 allele from S288c but not the one from Sigma can confer fMAPK pathway-independent transcription of FLO11. Biochemical analysis reveals differences in phosphorylation between the alleles. At the nucleotide level the two alleles differ in the number of tandem repeats in the ORF. A comparison of genomes between the two strains shows that many genes differ in size due to variation in repeat length.

Functional analysis with a barcoder yeast gene overexpression system.

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed "barFLEX." Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.

Global gene deletion analysis exploring yeast filamentous growth.

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.

Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology.

When grown on solid substrates, different microorganisms often form colonies with very specific morphologies. Whereas the pioneers of microbiology often used colony morphology to discriminate between species and strains, the phenomenon has not received much attention recently. In this study, we use a genome-wide assay in the model yeast Saccharomyces cerevisiae to identify all genes that affect colony morphology. We show that several major signalling cascades, including the MAPK, TORC, SNF1 and RIM101 pathways play a role, indicating that morphological changes are a reaction to changing environments. Other genes that affect colony morphology are involved in protein sorting and epigenetic regulation. Interestingly, the screen reveals only few genes that are likely to play a direct role in establishing colony morphology, with one notable example being FLO11, a gene encoding a cell-surface adhesin that has already been implicated in colony morphology, biofilm formation, and invasive and pseudohyphal growth. Using a series of modified promoters for fine-tuning FLO11 expression, we confirm the central role of Flo11 and show that differences in FLO11 expression result in distinct colony morphologies. Together, our results provide a first comprehensive look at the complex genetic network that underlies the diversity in the morphologies of yeast colonies.

Countries where HIV is concentrated among most-at-risk populations get disproportionally lower funding from PEPFAR.

The legislation reauthorizing the President's Emergency Plan for AIDS Relief (PEPFAR) in 2008 recognized the need for HIV/AIDS programs directed to most-at-risk populations, including men who have sex with men and people who inject drugs. To examine whether that goal is being met, we analyzed data from PEPFAR's Operational Plans for fiscal years 2009 and 2010. The eighteen countries in our study accounted for nearly two-thirds of overall PEPFAR financing for those fiscal years and approximately 60 percent of the total number of people living with HIV in the world in 2010. After controlling in each country for the number of people living with HIV, total population, and per capita income, we found that countries where HIV transmission occurs primarily among men who have sex with men and people who inject drugs received on average $235 million less in 2009 and 2010 than countries with widespread HIV epidemics among the general population. These findings raise questions about whether the country allocations of PEPFAR fully address needs based on the epidemiology of HIV infection in individual countries. Administrators should ensure that funding allocations directed to various countries reflect the best epidemiological data and latest science and best practices, and are devoid of bias against most-at-risk populations; they should also be more transparent about where PEPFAR's dollars go. Otherwise, it is unlikely that PEPFAR will realize its established goal of achieving an AIDS-free generation.

Vacuolar protein sorting genes regulate mat formation in Saccharomyces cerevisiae by Flo11p-dependent and -independent mechanisms.

Saccharomyces cerevisiae generates complex biofilms called mats on low-density (0.3%) agar plates. The mats can be morphologically divided into two regions: (i) hub, the interior region characterized by the presence of wrinkles and channels, and (ii) rim, the smooth periphery. Formation of mats depends on the adhesin Flo11p, which is also required for invasive growth, a phenotype in which the S. cerevisiae yeasts grow as chains of cells that dig into standard-density (2%) agar plates. Although both invasive growth and mat formation depend on Flo11p, mutations that perturb the multivesicular body (MVB) protein sorting pathway inhibit mat formation in a FLO11-independent manner. These mutants, represented by vps27Δ, disrupt mat formation but do not affect invasive growth, FLO11 gene or protein expression, or Flo11p localization. In contrast, an overlapping subset of MVB mutants (represented by ESCRT [endosomal sorting complex required for transport] complex genes such as VPS25) interrupt the Rim101p signal transduction cascade, which is required for FLO11 expression, and thus block both invasive growth and mat formation. In addition, this report shows that mature Flo11p is covalently associated with the cell wall and shed into the extracellular matrix of the growing mat.

Genotype to phenotype: a complex problem.

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.