A genetic switch for stable, long-term fermentative production of anabolic products in yeast

Amyris is the integrated renewable products company that is enabling the world’s leading brands to achieve sustainable growth. Amyris applies its innovative bioscience solutions to convert plant sugars into hydrocarbon molecules and produce specialty ingredients and consumer products. Production and marketing of the molecule farnesene (Biofene®) has already been commercialized with production scale in some markets. Farnesene has many applications as a renewable feedstock for polymers, nutraceuticals and cosmetics. To reduce the production cost of farnesene, at Amyris we engineer strains using a state-of-the art industrial synthetic biology platform to have high titer, yield, and productivity, and we perform fermentations in 200 m3 vessels over the course of many days or weeks. The challenge is that high-producer cells grow more slowly than spontaneous mutant low- or non-producer cells, especially in the nutrient-unlimited conditions of the seed train expansion, and yet must comprise the vast majority of the population. We have successfully addressed this challenge by developing an industrially-scalable genetic switch to successfully maintain high performance throughout lengthy fermentations. This genetic switch uses maltose (a cheap, non-toxic and metabolizable molecule) to control transcription such that when maltose is added in the seed train, product formation is shut off. This increased the growth of high-producer cells, resulting in higher inoculum purity and improved performance in bioreactors

Moving Beyond CHO: Alternative host systems may be the future of biotherapeutics

CHO cells are the primary expression system for recombinant proteins with significant investment over the last three decades resulting in robust cell lines and processes. The flexible nature of CHO has lent itself to multiple process formats, such as fed batch, perfusion and continuous cultures, and advances in omics technology has enabled customization of media formulations and targeted engineering of CHO cells. This knowledge has led to large gains in protein productivity that can be captured with culture duration and/or scale. Despite this, constant pressure exists to reduce cost of manufacturing and improve per batch productivity to meet the needs of increased patient populations and increase accessibility of these therapeutics. Biogen has partnered with MIT to take a holistic view of the potential future of biomanufacturing to identify technologies that can make step changes in productivity and cost reduction. This effort has identified the host system as the most important factor to enabling this vision. Specifically, a non-mammalian host could be the key to realizing the most significant gains in productivity and reduction in cost of manufacturing. Through this initiative, we sought to take a more comprehensive approach to investigate alternative hosts for recombinant antibody production. Eight non-mammalian hosts were selected based on several properties, including proven secretion of recombinant protein products, ability to glycosylate proteins, established genome or molecular biology toolkit, amongst others. The final panel of organisms included yeast, filamentous fungi, a diatom, and a trypanosome. In collaboration with Amyris, we evaluated these eight non-mammalian host cell lines to compare their suitability as a potential primary host for the biotechnology industry. Only non-engineered, wild-type strains were used as a starting point for this evaluation, which assessed the ability of each host to express the same IgG1 model antibody. The outcome of this comparative analysis demonstrated that several of the alternative hosts could express full length antibody with acceptable glycoforms. Additionally, the ease of culture, ability to engineer the genome, and flexibility of carbon source were assessed. As an output of this work, the most productive strains will be made available for use without restrictions to allow others in the community to freely work with these hosts. Based on this initial assessment, a strategy to further investigate the potential of the most promising hosts will be shared

A New Framework Combining Local-Region Division and Feature Selection for Micro-Expressions Recognition

Micro-expressions are deliberate or unconscious movements of people's psychological activities, reflecting the transient facial true expressions. Previous works focus on the whole face for micro-expressions recognition. These methods can extract a number of feature vectors which are relevant or irrelevant to the micro-expressions recognition. Besides, the high-dimension feature vectors can result in longer computational time and increased computational complexity. In order to address these problems, we propose a new framework which combines the local-region division and the feature selection. Based on the proposed framework, the original images can retain more efficient regions and filter out the invalid components of feature vectors. Specifically, with the joint efforts of the facial deformation identification model and facial action coding system, the global region is divided into seven local regions with their corresponding actions units. The ReliefF algorithm is used to select effective components of feature vectors and reduce the dimension. To evaluate the proposed framework, we conduct experiments on both the Chinese Academy of Sciences Micro-expression II Database and Spontaneous Micro-expression Database with Leave-One-Subject-Out Cross Validation method. The results show that the performance in local combined regions outperforms its counterpart in the global region, and the recognition accuracy is further improved with the combination of feature selection

Analysis of the expression and distribution of protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 in the normal adult mouse brain

IntroductionProtein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is crucial for the elongation of O-mannosyl glycans. Mutations in POMGNT1 cause muscle-eye-brain (MEB) disease, one of the main features of which is anatomical aberrations in the brain. A growing number of studies have shown that defects in POMGNT1 affect neuronal migration and distribution, disrupt basement membranes, and misalign Cajal-Retzius cells. Several studies have examined the distribution and expression of POMGNT1 in the fetal or neonatal brain for neurodevelopmental studies in the mouse or human brain. However, little is known about the neuroanatomical distribution and expression of POMGNT1 in the normal adult mouse brain.MethodsWe analyzed the expression of POMGNT1 mRNA and protein in the brains of various neuroanatomical regions and spinal cords by western blotting and RT-qPCR. We also detected the distribution profile of POMGnT1 in normal adult mouse brains by immunohistochemistry and double-immunofluorescence.ResultsIn the present study, we found that POMGNT1-positive cells were widely distributed in various regions of the brain, with high levels of expression in the cerebral cortex and hippocampus. In terms of cell type, POMGNT1 was predominantly expressed in neurons and was mainly enriched in glutamatergic neurons; to a lesser extent, it was expressed in glial cells. At the subcellular level, POMGNT1 was mainly co-localized with the Golgi apparatus, but expression in the endoplasmic reticulum and mitochondria could not be excluded.DiscussionThe present study suggests that POMGNT1, although widely expressed in various brain regions, may has some regional and cellular specificity, and the outcomes of this study provide a new laboratory basis for revealing the possible involvement of POMGNT1 in normal physiological functions of the brain from a morphological perspective

Antibody production in micro-organisms

Global demand for monoclonal antibody-based therapeutics (Mab’s) far exceeds current production capacity, and is expected to continue to grow based on current development pipelines. Despite their proven efficacy in a large number of indications, equitable use of these drugs is limited by the high cost of CHO-cell based production and purification. Micro-organisms such as yeasts and filamentous fungi present an attractive alternative for antibody production, but will require extensive genetic modification to achieve both high titers and mammalian-like glycosylation patterns in a secreted product that is easily purified. Towards this end, we developed state-of-the-art genetic engineering tools for eight micro-organisms to enable the highly efficient, targeted multiplexed integrations necessary for antibody production in these hosts. We demonstrated successful antibody production in several of these micro-organisms, paving the way to low-cost microbial fermentation to replace CHO fermentation

Smithian platform-bearing gondolellid conodonts from Yiwagou Section, northwestern China and implications for their geographic distribution in the Early Triassic

Abundant platform-bearing gondolellid conodonts, including Scythogondolella mosheri (Kozur and Mostler), Sc. phryna Orchard and Zonneveld, and Sc. cf. milleri (Müller), have been discovered from the Yiwagou Section of Tewo, together with Novispathodus waageni waageni (Sweet) and Nv. w. eowaageni Zhao and Orchard. This is the first report of Smithian platform-bearing gondolellids from the Paleo-Tethys region. In addition, Eurygnathodus costatus Staesche, E. hamadai(Koike), Parafurnishius xuanhanensis Yang et al., and the genera Pachycladina Staesche, Parachirognathus Clark, and Hadrodontina Staesche have also been recovered from Dienerian to Smithian strata at Yiwagou Section. Three conodont zones are established, in ascending order: Eurygnathodus costatus-E. hamadai Assemblage Zone, Novispathodus waageni-Scythogondolella mosheri Assemblage Zone, and the Pachycladina-Parachirognathus Assemblage Zone. The platform-bearing gondolellids were globally distributed just after the end-Permian mass extinction, but the formerly abundant Clarkina Kozur disappeared in the late Griesbachian. Platform-bearing gondolellids dramatically decreased to a minimum of diversity and extent in the Dienerian before recovering in the Smithian. Scythogondolella Kozur, probably a thermophilic and eurythermic genus, lived in all latitudes at this time whereas other genera did not cope with Smithian high temperatures and so became restricted to the high-latitude regions. However, the maximum temperature in the late Smithian likely caused the extinction of almost all platform-bearing gondolellids. Finally, the group returned to equatorial regions and achieved global distribution again in the cooler conditions of the late Spathian. We conclude that temperature (and to a lesser extent oxygen levels) exerted a strong control on the geographical distribution and evolution of platform-bearing gondolellids in the Early Triassic

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The first objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:CoA ligase (4CL) from Arabidopsis thaliana, and the chalcone synthase (CHS) from Hypericum androsaemum into Saccharomyces cerevisiae AH22 strain. Each gene was cloned and inserted into an expression vector under the control of separate individual GAL10 promoter. Besides PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase (TAL) activity, which enabled biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). The yeast AH22 strain co-expressing PAL, 4CL and CHS produced approximately 7 mg L-1 of naringenin and 0.8 mg L -1 of pinocembrin. Several byproducts, such as 2\u27,4\u27,6\u27-trihydroxydihydrochalcone and phloretin were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor L-tyrosine. The second objective was to synthesize flavones, apigenin and chrysin in yeast. Flavone synthase II (FNSII) from Gerbera hybrida, a plant cytochrome P450 monooxygenase, catalyzes naringenin to apigenin, liquiritigenin to 7,4\u27-dihydroxyflavone, and eriodictyol to luteolin. Our study is the first to show that FNSII is able to catalyze pinocembrin to chrysin, an anti-anxiety agent. Engineering the phenylpropanoid pathway from PAL, to FNSII enabled the production of apigenin from amino acid in yeast and the yield was 1.6 μM (0.3 μmol g CDW-1). Feeding studies showed that the production of apigenin was limited by aromatic precursors, L-phenylalanine and L-tyrosine, p-coumarate, and naringenin. Much significant higher yield of apigenin (27 μM or 9.2 pmol g CDW -1) was observed by feeding 400 μM of p-coumarate to the yeast co-expressing 4CL, CHS, and FNSII. The third objective was to synthesize novel flavonoids by feeding non-natural C4H substrate analogs to the yeast co-expressing C4H, 4CL, and CHS. Preliminary data have shown that 2-methylcinnamate, 2-fluorocinnamate, and 2-chlorocinnamate were converted to six novel flavonoids by the engineered pathway that have not been previously reported