30Antimicrobial-resistant infections are increasing across Asia. Aiming to evaluate antimicrobial access 31 and practices in Ho Chi Minh City (HCMC) of Vietnam, we mapped pharmacy locations and used a 32 simulated client method to calculate antimicrobial sales for paediatric diarrheal disease. We 33 additionally evaluated healthcare choices for parents and caregivers when their children experienced 34 diarrhoea. District 8 (population 396,175) of HCMC had 301 pharmacies (one for every 1,316 people), 35 with a density of 15.8 pharmacies/km 2 . A wide range of different treatments (n=57) were sold for 36 paediatric diarrheal disease, with 8% (3/37) and 22% (8/37) of the sampled pharmacies selling 37 antimicrobials for watery and mucoid diarrhoea, respectively. Despite the apparent abundance of 38 pharmacies, the majority of caregivers chose to take their child to a specialized hospital, with 81% 39 (319/396) and 88% (347/396) of responders selecting this as their first, second, or third choice for 40 watery and mucoid diarrhoea, respectively. Lastly, by combining denominators derived from 41 caregiver interviews and diarrheal incidence figures, we calculated that 16% (2,359/14,427) of watery 42 or mucoid diarrhoea episodes of the District 8 population aged 1 to <5 years would receive an 43 antimicrobial for diarrhoea annually, but antimicrobial prescribing was almost ten times greater in 44 hospitals than in the community. Our novel mixed-methods approach found that, whilst antimicrobials 45 are commonly available for paediatric diarrhoea in the community of HCMC, usage is greater in 46 hospitals. The observed non-standardized approach to diarrheal treatments is indicative of poor 47 recommendations. We advocate better guidelines, training and dissemination of information regarding 48 antimicrobials and their use in this location. 49 50 51 52 53 54 55 56 57 Committee (OxTREC approval 5110-16) and the local Institutional Review Board (IRB) of the 102 University of Medicine and Pharmacy at HCMC (No. 220/DHYD-HD). Written informed consent 103 was obtained from all participants over 18 years of age, or from parents and guardians if participant 104 was under 18 years. 105 106 Study setting and design 107With an estimated population of >8 million people, HCMC is the largest city in southern Vietnam 17 . 108

Abbreviations: IPP, isopentenyl diphosphate; MEP, 2-C-methyl-d-erythritol-4-phosphate; MVA, mevalonate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; G3P, glyceraldehyde-3-phosphate Introduction β-carotene, one of the carotenoid compounds, was found mainly in plants. Because of it's highly antioxidant activity, this compound was shown to have good effects to human health. It is the precursor of vitamin A which is critically required for human.1 Due to good health effects and yellow color, β-carotene is widely used in industries including nutraceuticals, pharmaceuticals, food colorants, cosmetics, and animal feed additives. However, production of natural β-carotene is not sufficient to afford demand of the market. Currently, more than 90% of commercially available β-carotene is chemically synthesized. 2This fact leads a number of research groups to the trend of seeking for alternative sources of natural β-carotene. Natural carotenogenic microorganisms, e.g. Blakeslea trispora, Rhodotorula glutinis, and Dunaliella salina have been used for fermentation to produce β-carotene. 3-5The availability of carotenoid genes from natural carotenogenic organisms leads to another strategy of natural carotenoid biosynthesis. Carotenoid genes were cloned and introduced into non-carotenogenic organism, Escherichia coli, for production of carotenoid, including β-carotene.6-19 E. coli is considered as one of the ideal hosts because of its convenience genetic engineering system and fast growth. However, to date, the production of β-carotene is still unable to support industrial needs because of insufficient yield and stability. Therefore, attempts are required for improvement of production of carotenoid in general and β-carotene in particular. Biosynthesis of β-carotene was affected by a number of factors. In our study, we focus in i. The different expression vector backbones ii. Origin of foreign genes iii. Availability and balance of isopentenyl diphosphate (IPP) which is the building blocks of carotenoids, and iv. Effect of additional carbon sources Biosynthesis partway of β-carotene is indicated in Figure 1. DMAPP and IPP, the building blocks of carotenoid are synthesized via 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway which is autonomous in our chosen host E. coli. However, in theory the natural yield of these precursors is only sufficient for natural need of E. coli in normal growth conditions which is far less than that required once the organism is used as a microbial factory to produce β-carotene. To address this issue, addition of the exogenous mevalonate (MVA) pathway was shown as a reasonable strategy. 6,12,18,20,21 The MVA pathway is divided into two portions, the upper (from acetyl-CoA to MVA) and the lower (from MVA to DMAPP and IPP). Natural E. coli harbors MEP pathway that enable biosynthesis of FPP from G3P and pyruvate, as well as IPP isomerase catalyze the two-way conversion of IPP and DMAPP, the precursors of carotenoid (yellow part). These precursors could ...