4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases—a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l⁻¹ in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.

Metabolic engineering is a powerful tool for the sustainable production of chemicals. Over the years, the exploration of microbial, animal and plant metabolism has generated a wealth of valuable genetic information. The prudent application of this knowledge on cellular metabolism and biochemistry has enabled the construction of novel metabolic pathways that do not exist in nature or enhance existing ones. The hand in hand development of computational technology, protein science and genetic manipulation tools has formed the basis of powerful emerging technologies that make the production of green chemicals and fuels a reality. Microbial production of chemicals is more feasible compared to plant and animal systems, due to simpler genetic make-up and amenable growth rates. Here, we summarize the recent progress in the synthesis of biofuels, value added chemicals, pharmaceuticals and nutraceuticals via metabolic engineering of microbes.

Aromatic polyketides have attractive biological activities and pharmacological properties. Different from other polyketides, aromatic polyketides are characterized by their polycyclic aromatic structure. The biosynthesis of aromatic polyketides is usually accomplished by the type II polyketide synthases (PKSs), which produce highly diverse polyketide chains by sequential condensation of the starter units with extender units, followed by reduction, cyclization, aromatization and tailoring reactions. Recently, significant progress has been made in characterization and engineering of type II PKSs to produce novel products and improve product titers. In this review, we briefly summarize the architectural organizations and genetic contributions of PKS genes to provide insight into the biosynthetic process. We then review the most recent progress in engineered biosynthesis of aromatic polyketides, with emphasis on generating novel molecular structures. We also discuss the current challenges and future perspectives in the rational engineering of type II PKSs for large scale production of aromatic polyketides.

Microbial coculture engineering has emerged as a promising strategy for biomanufacturing. Stability and self-regulation pose a significant challenge for the generation of intrinsically robust cocultures for large-scale applications. Here, we introduce the use of multi-metabolite cross-feeding (MMCF) to establish a close correlation between the strains and the design rules for selecting the appropriate metabolic branches. This leads to an intrinicially stable two-strain coculture where the population composition and the product titer are insensitive to the initial inoculation ratios. With an intermediate-responsive biosensor, the population of the microbial coculture is autonomously balanced to minimize intermediate accumulation. This static-dynamic strategy is extendable to three-strain cocultures, as demonstrated with de novo biosynthesis of silybin/isosilybin. This strategy is generally applicable, paving the way to the industrial application of microbial cocultures.

Quorum sensing (QS) is a ubiquitous cell–cell communication mechanism that can be employed to autonomously and dynamically control metabolic fluxes. However, since the functions of genetic components in the circuits are not fully understood, the developed QS circuits are still less sophisticated for regulating multiple sets of genes or operons in metabolic engineering applications. Here, we discover the regulatory roles of a CRP-binding site and the lux box to −10 region within luxR-luxI intergenic sequence in controlling the lux-type QS promoters. By varying the numbers of the CRP-binding site and redesigning the lux box to −10 site sequence, we create a library of QS variants that possess both high dynamic ranges and low leakiness. These circuits are successfully applied to achieve diverse metabolic control in salicylic acid and 4-hydroxycoumarin biosynthetic pathways in Escherichia coli. This work expands the toolbox for dynamic control of multiple metabolic fluxes under complex metabolic background and presents paradigms to engineer metabolic pathways for high-level synthesis of target products.