Non-Saccharomyces yeasts for beer production: Insights into safety aspects and considerations.

The application of non-Saccharomyces yeasts in beer as a natural tool for innovation, to create different aroma profiles and flavoured non-alcoholic beers, has attracted great interest from both researchers and commercial brewers. As a result, a higher diversity of non-Saccharomyces yeasts for beer production is expected on the market in the coming years. However, the safe use of non-Saccharomyces yeasts has not been broadly investigated and no guidance for the safety assessment of yeasts is published. The fundamentals of a safety assessment include an accurate taxonomic species identification using up-to date methods, along with a literature study regarding the yeast species in question. The strain-specific safety concerns that should be assessed involve pathogenic potential, antifungal resistance, production of biogenic amines and possible allergic reactions. However, yeast safety assessment is in its infancy compared to bacterial safety assessment and research is needed to set cut-off values for antifungal resistance, identify potential virulence genes and validate screening tools to assess yeast strains. Finally, the individual breweries are responsible for the safety related to the process in which yeasts are applied and throughout the shelf life of the beer. The application of non-Saccharomyces yeasts for industrial beer production is promising in terms of defining new prototypes and developing healthier and safer beers, but only if good food safety measures, i.e., both for the strain and the production process, are in place throughout the food value chain. In this way, the ancient role of yeasts in making beverages safer and thereby improving food safety is emphasized.

Designing microorganisms for heterologous biosynthesis of cannabinoids.

During the last decade, the use of medical Cannabis has expanded globally and legislation is getting more liberal in many countries, facilitating the research on cannabinoids. The unique interaction of cannabinoids with the human endocannabinoid system makes these compounds an interesting target to be studied as therapeutic agents for the treatment of several medical conditions. However, currently there are important limitations in the study, production and use of cannabinoids as pharmaceutical drugs. Besides the main constituent tetrahydrocannabinolic acid, the structurally related compound cannabidiol is of high interest as drug candidate. From the more than 100 known cannabinoids reported, most can only be extracted in very low amounts and their pharmacological profile has not been determined. Today, cannabinoids are isolated from the strictly regulated Cannabis plant, and the supply of compounds with sufficient quality is a major problem. Biotechnological production could be an attractive alternative mode of production. Herein, we explore the potential use of synthetic biology as an alternative strategy for synthesis of cannabinoids in heterologous hosts. We summarize the current knowledge surrounding cannabinoids biosynthesis and present a comprehensive description of the key steps of the genuine and artificial pathway, systems biotechnology needs and platform optimization.

Microbial production of next-generation stevia sweeteners.

BACKGROUND: The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana. RESULTS: In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1. CONCLUSIONS: Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.

Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C6 glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (T(m)), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3'-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/.

Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms.

Terpenoids represent a diverse class of molecules that provide a wealth of opportunities to address many human health and societal issues. The expansive array of structures and functionalities that have been evolved in nature provide an excellent pool of molecules for use in human therapeutics. While this class of molecules has members with therapeutic properties including anticancer, antiparasitic, antimicrobial, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties, supply limitations prevent the large scale use of some molecules. Many of these molecules are only found in ppm levels in nature thus requiring massive harvesting to obtain sufficient amounts of the drug. Synthetic biology and metabolic engineering provide innovative approaches to increase the production of the desired molecule in the native organism, and most importantly, transfer the biosynthetic pathways to other hosts. Microbial systems are well studied, and genetic manipulations allow the optimization of microbial metabolisms for the production of common terpenoid precursors. Using a host of tools, unprecedented advancements in the large scale production of terpenoids have been achieved in recent years. Identification of limiting steps and pathway regulation, coupled with design strategies to minimize terpenoid byproducts wih a high flux to the desired biosynthetic pathways, have yielded greater than 100-fold improvements in the production of a range of terpenoids. This review focuses on the biodiversity of terpenoids, the biosynthetic pathways involved, and engineering efforts to maximize the production through these pathways.