In modern societies, conscious consumers demand healthy, fresh and natural foods devoid of added chemical preservatives and stabilizers. The use of lactic acid bacteria (LAB) to preserve food is one of the oldest and best characterized approach. Production of organic acids is the main feature LAB use to outcompete spoilage organisms, but several other mechanisms have been demonstrated. In this review, a critical overview of the mechanisms used by LAB to inhibit spoilage organisms will be presented. Discrepancies between the concentrations of compounds resulting from LAB activity and their inhibitory amounts are discussed. Technical limitations hindering discoveries in this field as well as future trends in the application of LAB solutions to food bioprotection will be covered, including antifungal peptides and competitive exclusion.

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p -coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p -coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus , a bacterial-type enzyme from the amoeba Dictyostelium discoideum , and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD ₆₀₀ ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p -coumaric acid in Escherichia coli (producing 440 μM p -coumaric acid OD ₆₀₀ unit ⁻¹ in the medium) and in Lactococcus lactis . The enzymes were also efficient in Saccharomyces cerevisiae , where p -coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is inactivated by quercetin and kaempferol. Both biosensors showed over a 7-fold increase of the fluorescent signal after addition of their specific effectors, and a linear correlation between the fluorescence intensity and externally added flavonoid concentration. The QdoR-biosensor was successfully applied for detection of kaempferol production in vivo at the single cell level by fluorescence-activated cell sorting. Furthermore, the amount of kaempferol produced highly correlated with the specific fluorescence of E. coli cells containing a flavonol synthase from Arabidopsis thaliana (fls1). We expect the designed biosensors to be applied for isolation of genes involved in flavonoid biosynthetic pathways.