Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule known to alter receptor binding affinity and potency. We also demonstrated that our biological system can be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids, which will allow for more rigorous study of cannabinoids and ultimately the development of new treatments for a variety of human ailments. We initiated construction of the cannabinoid-producing yeast by focusing first on production of OA , an initial intermediate in the cannabinoid biosynthetic pathway. Two Cannabis enzymes, a tetraketide synthase and an olivetolic acid cyclase ,6 have been reported to produce OA from hexanoyl-CoA and malonyl-CoA. To produce OA in yeast, we introduced a CsTKS and CsOAC expression cassette into S. cerevisiae to generate strain yCAN01 . The strain produced 0.2 mg/L OA from galactose , consistent with the fact that S. cerevisiae maintains low intracellular levels of hexanoyl-CoA. To increase the supply of hexanoyl-CoA, we fed 1 mM hexanoic acid, which can be converted to hexanoyl-CoA by an endogenous acyl activating enzyme , and observed six-fold higher OA production compared to no exogenous hexanoic acid feeding. A known byproduct of TKS, hexanoyl triacetic acid lactone ,6 was also detected . To optimize the conversion of hexanoic acid to hexanoyl-CoA, we introduced into yCAN01 an AAE from Cannabis , which is thought to catalyze this step in planta. The resulting strain showed a two-fold increase in OA titer when fed 1 mM hexanoic acid . To produce hexanoyl-CoA from galactose and complete the OA pathway, we introduced into yCAN01 a previously reported hexanoyl-CoA pathway.
The resulting strain produced 1.6 mg/L OA . CBGA, the precursor to THCA, CBDA, and numerous other cannabinoids, how to dry cannabis is produced from the mevalonate pathway intermediate GPP and OA by GOT. GOT activity was detected in Cannabis extracts over two decades ago, and a Cannabis GOT was patented ten years later. To enable in vivo testing of CsPT1, we constructed a GPP-over producing strain with an upregulated mevalonate pathway and a mutant gene of the endogenous farnesyl pyrophosphate synthase ERG, which preferentially produces GPP over FPP.However, we were unable to observe any GOT activity when we expressed CsPT1 or truncations thereof in yCAN10. To identify an enzyme with GOT activity that would function in yeast, we searched for candidate prenyltransferase enzymes from Cannabis and other organisms. These included NphB, a soluble PT from Streptomyces sp. with GOT activity in vitro, as well as HlPT1L and HlPT2, two PTs involved in bitter acid biosynthesis in Humulus lupulus, a close relative of Cannabis. In addition, we mined published Cannabis transcriptomes for GOT candidates.We set out to establish a biosynthetic approach for the production of this class of cannabinoid analogues from different fatty acids, hypothesizing that the observed promiscuity of our pathway towards butanoylCoA would translate to other precursors . To probe the analogue production capability of our engineered strains, we fed yCAN31 an array of 19 different fatty acids with various chain lengths, branching and degrees of saturation . LC-MS analysis revealed the production of OA and CBGA analogues from pentanoic acid , heptanoic acid , 4-methylhexanoic acid , 5-hexenoic acid and 6-heptynoic acid . Subsequent supplementation of yCAN40 with this subset of fatty acids yielded the respective THCA analogues . Furthermore, the functionalization of the pharmacophore with an alkene or alkyne terminal group enabled simple post-fermentation modification and thus the construction of side chains intractable to direct incorporation. As proof of concept, we performed copper-catalyzed azide-alkyne cycloaddition on the respective 6- heptynoic acid CBGA as well as THCA analogues with an azide-PEG3-biotin conjugate.
The corresponding products were detected by LC-MS demonstrating that the accessible chemical space of our process can be further expanded. Our results illustrate a novel avenue towards the production of cannabinoid analogues with tailored C3 sidechains. In summary, we engineered yeast strains capable of producing the major cannabinoids found in Cannabis from galactose. Pending the identification of novel cannabinoid synthases, we expect to be able to produce a large fraction of this class of natural molecules. Additionally, we further expanded the chemical space of cannabinoids by establishing and harnessing the intrinsic promiscuity of the cannabinoid pathway to produce unnatural cannabinoids including molecules with side groups amenable to further chemical derivatization. This work lays the foundation for the large-scale fermentation of cannabinoids, independent of Cannabis cultivation, which will enable the pharmacological study of these highly promising compounds and ultimately new and better medicines.Concerns about global climate change, energy security, and unstable fuel prices have caused many decision makers and policy experts worldwide to closely examine the need for more sustainable transportation strategies. Sustainable strategies include clean fuels, vehicle technologies, transportation demand management, and integrated land use and transportation strategies . Bikesharing—the shared use of a bicycle fleet—is one mobility strategy that could help address many of these concerns. In recent years, interest in this evolving concept has spread across the globe. At present, there are an estimated 100 programs in approximately 125 cities around the world with over 139,300 bicycles on four continents and another 45 planned in 22 nations in 2010. Despite rapid global motorization, worldwide bicycle use has generally increased over the past 30 years. Indeed, bicycling in Dutch, German, and Danish cities increased between 20 to 43% between 1975 and 1995 . While cycling growth and trends vary worldwide, bike sharing offers a transportation alternative to increase bicycle use by integrating cycling into the transportation system and making it more convenient and attractive to users. The principle of bike sharing is simple.
Individuals use bicycles on an “as-needed” basis without the costs and responsibilities of bike ownership. Bike sharing is short-term bicycle access, which provides its users with an environmentally friendly form of public transportation. This flexible short-term usage scheme targets daily mobility and allows users to access public bicycles at unattended bike stations. Bicycle reservations, pick-up, and drop-off are self-service. Commonly concentrated in urban settings, bike sharing programs also provide multiple bike station locations that enable users to pick up and return bicycles to different stations. Bike sharing programs typically cover bicycle purchase and maintenance costs, as well as storage and parking responsibilities . Besides individual user perks, bike sharing also offers environmental, social, and transportation-related benefits. For instance, bike sharing provides a low-carbon solution to the “last mile” problem. The “last mile” refers to the short distance between home and public transit and/or transit stations and the workplace, which may be too far to walk. Thus, bike sharing has the potential to play an important role in bridging the gap in existing transportation networks, as well as encouraging individuals to use multiple transportation modes. Potential bike sharing benefits include: 1) increased mobility options; 2) cost savings from modal shifts; 3) lower implementation and operational costs ; 4) reduced traffic congestion; 5) reduced fuel use; 6) increased use of public transit and alternative modes ; 7) increased health benefits; and 8) greater environmental awareness. The ultimate goal of bike sharing is to expand and integrate cycling into transportation systems, so that it can more readily become a daily transportation mode. In recent years, bike sharing also has expanded to college and work campuses throughout North America. Indeed, there are over 65 college/university bike sharing programs operating throughout North America and another 10 programs planned in 2010. Examples of college/university programs worldwide include “CibiUAM” at the Universidad Autonoma de Madrid in Spain and “Velocampus Leeds” at the University of Leeds in the United Kingdom . The focus of this paper, however, is on citywide systems that are open to residents and visitors, as opposed to closed systems that are only accessible to students and employees of a university or major employer. Furthermore, the authors do not address bike rental programs, best way to dry cannabis which also have expanded worldwide. Unlike bike sharing, bike rental traditionally targets users interested in leisure-oriented mobility and are most prevalent in areas with a high tourist concentration. Bike rental systems generally consist of a single or limited number of bike stations that are operated by a service attendant. A majority of bike rental programs also require users to return rented bicycles to the original bike station and are generally operated on an hourly pricing basis. Over the last 43 years, bike sharing’s evolution has been categorized into three key phases . These include the first generation, called “White Bikes” ; the second generation: “Coin-Deposit Systems;” and the third generation or “Information Technology -Based Systems” . In this paper, the authors propose a fourth generation, called: “Demand-Responsive, Multi-Modal Systems,” which builds upon the third. This paper is organized into seven sections. First, the authors present a history of bike sharing in Europe, the Americas, and Asia, focused upon the first two generations. Next, current bike sharing activities are discussed in Europe, the Americas, and Asia. Third, bike sharing business models and vendors are described. Next, the authors summarize the current understanding of the social and environmental benefits associated with bike sharing. Fifth, lessons learned are presented. Next, a fourth bike sharing generation is proposed with an eye toward future developments and innovation.
Finally, the authors conclude with a summary and recommendations for future bike sharing research.Despite earlier experiences, the bike sharing concept caught on and led to the first generation of bike sharing known as “White Bikes” . In a free bike sharing system, the bicycle is the main program component. Other distinguishing characteristics of first generation bike sharing include that bicycles were usually painted one bright color, unlocked, and placed haphazardly throughout an area for free use. Other cities that implemented a free bike system were La Rochelle, France in 1974 and Cambridge in the UK in 1993, called “Green Bike Schemes.” Soon after Green Bike Scheme’s launch, the almost 300 shared bicycles in Cambridge were stolen, resulting in program failure . However, the La Rochelle initiative, called “Vélos Jaunes” or “Yellow Bikes,” proved to be successful and continues to operate today. La Rochelle’s Mayor, Michel Crépeau, created Vélos Jaunes. Similar to Amsterdam’s White Bike Plan, Vélos Jaunes was launched as an environmentally progressive measure. Through the strong support of La Rochelle’s Urban Community, Vélos Jaunes became the first successful bike sharing program in France. Problems with Free Bike Systems led the city government and the City Bike Foundation of Copenhagen, Denmark to launch a bike sharing service that was different from any previous system. In January 1995, “Bycyken” was launched as the first large-scale urban bike sharing program in Europe. This initiative included 1,100 specially designed bicycles that were locked and placed throughout downtown Copenhagen at designated city bike racks . Bicycles were unlocked with a 20 DKK coin deposit that was refunded upon bicycle return. Bycyken of Copenhagen is famous not only because it continues to operate with more than 2,000 bicycles and 110 city bike racks today but also because it led to the second generation of bike sharing, known as “Coin-Deposit Systems.” The main components of this generation are: 1) distinguishable bicycles ; 2) designated docking stations in which bikes can be locked, borrowed, and returned; and 3) small deposits to unlock the bikes. Soon after the implementation of coin-deposit systems, the Copenhagen model led to a series of European bike sharing programs including: “Bycykler” in Sandnes, Norway ; “City Bikes” in Helsinki, Finland ; and “Bycykel” in Arhus, Denmark . The experience of these coin-deposit systems demonstrated that second-generation systems were more expensive to operate than early systems. In many cases, local governments also provided bike sharing organizations with funding. The incorporation of designated bicycle stations and the use of coin-deposit locks in second-generation systems created a much more reliable bike sharing system that was both dependable and more theft resistant. While amounts vary by country, coin deposit fees are generally low . Also, these systems do not issue a time limit for bicycle use, which means that bikes are often used for long time periods or not returned at all. The major problem with coin-deposit systems is bicycle theft, which can be attributed to customer anonymity.