Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production

Since the advent of systems and synthetic biology, many studies have sought to harness microbes as cell factories through genetic and metabolic engineering approaches. Yeast and filamentous fungi have been successfully harnessed to produce fine and high value-added chemical products. In this review, we present some of the most promising advances from recent years in the use of fungi for this purpose, focusing on the manipulation of fungal strains using systems and synthetic biology tools to improve metabolic flow and the flow of secondary metabolites by pathway redesign. We also review the roles of bioinformatics analysis and predictions in synthetic circuits, highlighting in silico systemic approaches to improve the efficiency of synthetic modules

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.Fil: Monteiro, Lummy Maria Oliveira. Universidade de Sao Paulo; BrasilFil: Arruda, Leticia Magalhães. Universidade de Sao Paulo; BrasilFil: Sanches Medeiros, Ananda. Universidade de Sao Paulo; BrasilFil: Martins Santana, Leonardo. Universidade de Sao Paulo; BrasilFil: Alves, Luana de Fátima. Universidade de Sao Paulo; BrasilFil: Defelipe, Lucas Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Turjanski, Adrian Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guazzaroni, Mara Eugenia. Universidade de Sao Paulo; BrasilFil: de Lorenzo, Victor. Consejo Superior de Investigaciones Científicas. Centro Nacional de Biotecnología; EspañaFil: Silva Rocha, Rafael. Universidade de Sao Paulo; Brasi

Acessando a regulação transcricional da formação de biofilme em Escherichia coli utilizando bibliotecas genômica e de promotores

Biofilms are complex structures formed by bacterial communities of the same or different species, embedded in an extracellular matrix composed of polymeric extracellular substances (EPS), such as polysaccharides, proteins (as curli structures), and nucleic acids. The regulatory networks of biofilm formation are composed of the integration of environmental and intracellular stimuli. One of its layers is transcriptional regulation, which is capable of drastically altering bacterial gene expression, converting it from a free-living style to a sessile behavior. To better understand how biofilm-related genes are regulated by different transcription factors, we aimed to apply the SortSeq technique to deeply investigate the architecture of central promoters of biofilm formation. We started this work with 21 promoters of interest, associated directly with biofilm formation and motility. A library containing these 21 promoters mutated randomly at a 10% rate was constructed and tested. The preliminary tests showed that this library had too many mutations and truncations per promoter sequence, and few promoter variants, making unfeasible the acquisition of results with it. This way, we decided to focus on two main promoters of genes for biofilm formation, related to curli synthesis: csgBAp, and csgDp. Their promoters are restricted to the same intergenic region between their genes and have a strict relation, once csgD is a master regulator of transcription of the csgBA operon. We divided this intergenic region into four parts and built libraries with region-focused random mutations. These four libraries were individually inserted into the original promoters, which were assembled in a low-copy number plasmid and modulate the expression of sfgfp and mCherry reporter genes (csgBD-reporter plasmid). To access how the promoter mutations could affect the expression of each gene, we transformed the four libraries into the E. coli W3110 RpoS+ and cultivated it to the stationary phase. Reproducing the SortSeq technique, the cultures were analyzed by flow cytometry and sorted into four tubes according to their fluorescence for sfGFP, mCherry, sfGFP, and mCherry, and Negative (no expression for both reporters). Triplicates of this experiment were mini-prepped and deep-sequenced using Illumina platform. This new library design and SortSeq approach allowed us to better understand how each promoter region can affect the expression of both genes individually and at the same time, helping us to confirm or deny binding sites already predicted. We also found new possible binding sites for transcription factors not described yet, which were used for the prediction of new regulators to these promoters. This library gave us some insights into the logic involved in csgB bistability regulation. To expand the investigation of csgBA and csgD promoters we constructed a genomic library of the E. coli W3110 RpoS+ strain, using a barcoded-Tn5 plasmid. The Tn5 transposase present in the plasmid inserts randomly a unique barcode in a unique region of the genomic DNA. Then, we map the barcode to its corresponding genomic region, allowing the mutated site identification only by sequencing the barcode. We transformed our csgBD-reporter plasmid (without any library insertion) into it and used the SortSeq approach to sort the bacteria in the same four phenotypes and sequence the inserted barcode through the NGS technique. This second approach led to the identification of new players in the gene regulation of these important genes associated with biofilm formation. Combining these two types of libraries, we had new insights about the csgBA and csgD promoters\' regulation that will be tested and confirmed in the future.Biofilmes são estruturas complexas formadas por comunidades bacterianas da mesma ou de espécies diferentes, embebidas em uma matriz extracelular composta por substâncias extracelulares poliméricas (EPS), como polissacarídeos, proteínas (como estruturas curli) e ácidos nucléicos. As redes reguladoras da formação do biofilme são compostas pela integração de estímulos ambientais e intracelulares. Uma de suas camadas é a regulação transcricional, que é capaz de alterar drasticamente a expressão gênica bacteriana, convertendo-a de um estilo de vida livre para um comportamento séssil. Para entender melhor como os genes relacionados ao biofilme são regulados por diferentes fatores de transcrição, decidimos aplicar a técnica de SortSeq para investigar a fundo a arquitetura de promotores centrais relacionados à formação de biofilme. Iniciamos este trabalho com 21 promotores, associados diretamente ao desenvolvimento do biofilme e motilidade bacteriana. Construímos e testamos uma biblioteca contendo esses 21 promotores mutados aleatoriamente a uma taxa de 10%. Os testes preliminares mostraram que esta biblioteca possuía muitas mutações e truncamentos das sequências promotoras, e poucas variantes, inviabilizando a aquisição de resultados com ela. Desta forma, decidimos focar em dois principais promotores de genes relacionados à síntese de curli: csgBAp e csgDp. Seus promotores estão restritos a uma mesma região intergênica e possuem uma relação estrita, uma vez que CsgD é um regulador mestre da transcrição do operon csgBA. Dividimos essa região intergênica em quatro partes e construímos bibliotecas com mutações focalizadas e aleatórias. Essas quatro bibliotecas foram inseridas individualmente nos promotores originais, os quais já haviam sido clonados em um plasmídeo de baixo número de cópias e modulam a expressão dos genes repórteres sfgfp e mCherry (plasmídeo csgBD-repórter). Para acessar como as mutações do promotor podem afetar a expressão de cada gene, transformamos as quatro bibliotecas na linhagem de E. coli W3110 RpoS+ e as cultivamos até a fase estacionária. Reproduzindo a técnica SortSeq, as culturas foram analisadas por citometria de fluxo e classificadas em quatro tubos de acordo com sua fluorescência para sfGFP, mCherry, sfGFP e mCherry, e negativo (sem expressão para ambos os repórteres). Triplicatas deste experimento tiveram seus plasmídeos extraídos e sequenciados usando a plataforma Illumina. Este novo design de biblioteca e abordagem SortSeq nos permitiu entender melhor como cada região promotora pode afetar a expressão de ambos os genes individual e conjuntamente, ajudando-nos a confirmar ou negar a atuação de sítios de ligação a fatores de transcrição já descritos. Também encontramos novos possíveis sítios de ligação para fatores de transcrição ainda não descritos, os quais foram utilizados para a predição de novos reguladores para esses promotores. Para expandir a investigação dos promotores csgBA e csgD construímos uma biblioteca genômica da cepa E. coli W3110 RpoS+, usando um plasmídeo contendo a transposase Tn5 e sequencias de DNA como código de barras. A transposase Tn5 presente no plasmídeo insere aleatoriamente um código de barras único em uma região única do DNA genômico. Em seguida, mapeamos o código de barras para sua região genômica correspondente, permitindo a identificação do sítio mutado apenas pelo sequenciamento do código de barras. Transformamos nosso plasmídeo csgBD-reporter (sem qualquer inserção de biblioteca) na biblioteca e usamos a abordagem SortSeq para dividir as bactérias nos mesmos quatro fenótipos e sequenciar o código de barras inserido por meio da técnica NGS. Esta segunda abordagem levou à identificação de novos atores na regulação gênica desses genes tão importantes associados à formação do biofilme. Combinando esses dois tipos de bibliotecas, descobrimos possíveis novas regulações dos promotores de csgBA e csgD que serão testados e confirmados futuramente

Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli

The engineering of synthetic circuits in cells relies on the use of well-characterized biological parts that would perform predicted functions under the situation considered, and many efforts have been taken to set biological standards that could define the basic features of these parts. However, since most synthetic biology projects usually require a particular cellular chassis and set of growth conditions, defining standards in the field is not a simple task as gene expression measurements could be affected severely by genetic background and culture conditions. In this study, we addressed promoter parameterization in bacteria in different genetic backgrounds and growth conditions. We found that a small set of constitutive promoters of different strengths controlling a short-lived GFP reporter placed in a low-copy number plasmid produces remarkably reproducible results that allow for the calibration of promoter activity over different genetic backgrounds and physiological conditions, thus providing a simple way to set standards of promoter activity in bacteria. Based on these results, we proposed the utilization of synthetic constitutive promoters as tools for calibration for the standardization of biological parts, in a way similar to the use of DNA and protein ladders in molecular biology as references for comparison with samples of interest

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.R.S.-R. and M.-E.G. were supported by Young Research Awards, grant nos. 2012/22921-8 and 2015/04309-1, São Paulo Research Foundation (FAPESP). L.M.O.M., A.S.-M., L.M.-S., and L.F.A. were supported by FAPESP Ph.D. Fellowships (grant nos. 2016/19179-9, 2016/06323-4, 2017/17924-1, and 2018/04810-0)