Application of whole-cell biosensors for analysis and improvement of L- and D-lactic acid fermentation by Lactobacillus spp. from the waste of glucose syrup production.

BACKGROUND: Lactic acid is one of the most important organic acids, with various applications in the food, beverage, pharmaceutical, cosmetic, and chemical industries. Optically pure forms of L- and D-lactic acid produced via microbial fermentation play an important role in the synthesis of biodegradable polylactic acid. Alternative substrates, including by-products and residues from the agro-food industry, provide a cost-effective solution for lactic acid production and are a promising avenue for the circular economy. RESULTS: In this study, the transcription factor (TF)-based whole-cell biosensor strategy was developed for the L- and D-lactic acid determination. It was cross validated with commonly used high-performance liquid chromatography and enzymatic methods. The utility of biosensors as an efficient analytical tool was demonstrated by their application for the lactic acid determination and fermentation improvement. We explored the ability of Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis, and Lactobacillus amylovorus to biosynthesize optically pure L-lactic acid, D-lactic acid or mixture of both from organic-rich residual fraction (ORRF), a waste of glucose syrup production from wheat starch. The fermentation of this complex industrial waste allowed the production of lactic acid without additional pretreatment obtaining yields from 0.5 to 0.9 Cmol/Cmol glucose. CONCLUSIONS: This study highlights the utility of whole cell biosensors for the determination of L- and D-forms of lactic acid. The fermentation of L-lactic acid, D-lactic acid and mixture of both by L. paracasei, L. lactis, and L. amylovorus, respectively, was demonstrated using waste of glucose syrup production, the ORRF.

Transcription factor-based biosensors for detection of naturally occurring phenolic acids.

Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli, Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p-hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism.

Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440.

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/PPP_RS00425 from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/PPP_RS00425 is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.

Identification and characterization of L- and D-lactate-inducible systems from Escherichia coli MG1655, Cupriavidus necator H16 and Pseudomonas species.

Lactic acid is an important platform chemical used for the production of various compounds including polylactic acid (PLA). Optically pure L- and D-lactic acids are required to obtain high quality PLA. To advance the development and selection of microbial strains for improved production of lactic acid enantiomers, a high-throughput screening, dynamic pathway control, or real-time monitoring are often applied. Inducible gene expression systems and their application in the genetically encoded biosensors contribute to the development of these techniques and are important devices for the advancement of lactic acid biotechnology. Here, we identify and characterize eleven lactate-inducible systems from Escherichia coli, Cupriavidus necator, and Pseudomonas spp. The specificity and dynamics of these systems in response to L- and D-lactate, or structurally similar compounds are investigated. We demonstrate that the inducible systems EcLldR/PlldP and CnGntR/PH16_RS19190 respond only to the L-lactate, exhibiting approximately 19- and 24-fold induction, respectively. Despite neither of the examined bacteria possess the D-lactate-specific inducible system, the PaPdhR/PlldP and PfPdhR/PlldP are induced approximately 37- and 366-fold, respectively, by D-lactate and can be used for developing biosensor with improved specificity. The findings of this study provide an insight into understanding of L- and D-lactate-inducible systems that can be employed as sensing and tuneable devices in synthetic biology.

Bioproduction of l- and d-lactic acids: advances and trends in microbial strain application and engineering.

Lactic acid is an important platform chemical used in the food, agriculture, cosmetic, pharmaceutical, and chemical industries. It serves as a building block for the production of polylactic acid (PLA), a biodegradable polymer, which can replace traditional petroleum-based plastics and help to reduce environmental pollution. Cost-effective production of optically pure l- and d-lactic acids is necessary to achieve a quality and thermostable PLA product. This paper evaluates research advances in the bioproduction of l- and d-lactic acids using microbial fermentation. Special emphasis is given to the development of metabolically engineered microbial strains and processes tailored to alternative and flexible feedstock concepts such as: lignocellulose, glycerol, C1-gases, and agricultural-food industry byproducts. Alternative fermentation concepts that can improve lactic acid production are discussed. The potential use of inducible gene expression systems for the development of biosensors to facilitate the screening and engineering of lactic acid-producing microorganisms is discussed.

Valorization of Bilberry (Vaccinium myrtillus L.) Pomace by Enzyme-Assisted Extraction: Process Optimization and Comparison with Conventional Solid-Liquid Extraction.

Bilberry (Vaccinium myrtillus L.) pomace contains a significant amount of polyphenols and can serve as a basis for food additives, nutraceuticals, and functional foods. Although various techniques can be employed to recover bioactive fractions from berry pomaces, data on enzyme-assisted extraction (EAE) of bilberry pomace are rather scarce. This study aimed to optimize critical EAE parameters using Viscozyme L to obtain a high-yield extract with enhanced antioxidant capacity. Central composite design and response surface methodology evaluating the effect of four independent variables, namely, pH, temperature, extraction time, and enzyme concentration on three responses, were employed to define optimal EAE conditions. Under the optimal conditions (pH: 4.5, temperature 46 °C, 1 h of extraction, and 2 active units (AU) of Viscozyme L/g of pomace), EAE yielded 56.15 g/100 g DW of the water-soluble fraction. Comparison with conventional maceration indicated that EAE, besides the yield, significantly increased the in vitro antioxidant capacity measured by the total phenolic content, ABTS, ORAC, and CUPRAC assays. Moreover, an increase was observed for the measured mono- and disaccharide as well as anthocyanin content. Overall, this study demonstrates the improved efficiency of EAE over conventional solid-liquid extraction to recover fractions with a higher yield and enhanced functional properties in a fast and sustainable manner.

Advances and Prospects of Phenolic Acids Production, Biorefinery and Analysis.

Biotechnological production of phenolic acids is attracting increased interest due to their superior antioxidant activity, as well as other antimicrobial, dietary, and health benefits. As secondary metabolites, primarily found in plants and fungi, they are effective free radical scavengers due to the phenolic group available in their structure. Therefore, phenolic acids are widely utilised by pharmaceutical, food, cosmetic, and chemical industries. A demand for phenolic acids is mostly satisfied by utilising chemically synthesised compounds, with only a low quantity obtained from natural sources. As an alternative to chemical synthesis, environmentally friendly bio-based technologies are necessary for development in large-scale production. One of the most promising sustainable technologies is the utilisation of microbial cell factories for biosynthesis of phenolic acids. In this paper, we perform a systematic comparison of the best known natural sources of phenolic acids. The advances and prospects in the development of microbial cell factories for biosynthesis of these bioactive compounds are discussed in more detail. A special consideration is given to the modern production methods and analytics of phenolic acids.