During microbial applications, metabolic burdens can lead to a significant drop in cell performance. Novel synthetic biology tools or multi-step bioprocessing (e.g., fermentation followed by chemical conversions) are therefore needed to avoid compromised biochemical productivity from over-burdened cells. A possible solution to address metabolic burden is Division of Labor (DoL) via natural and synthetic microbial consortia. In particular, consolidated bioprocesses and metabolic cooperation for detoxification or cross feeding (e.g., vitamin C fermentation) have shown numerous successes in industrial level applications. However, distributing a metabolic pathway among proper hosts remains an engineering conundrum due to several challenges: complex subpopulation dynamics/interactions with a short time-window for stable production, suboptimal cultivation of microbial communities, proliferation of cheaters or low-producers, intermediate metabolite dilution, transport barriers between species, and breaks in metabolite channeling through biosynthesis pathways. To develop stable consortia, optimization of strain inoculations, nutritional divergence and crossing feeding, evolution of mutualistic growth, cell immobilization, and biosensors may potentially be used to control cell populations. Another opportunity is direct integration of non-bioprocesses (e.g., microbial electrosynthesis) to power cell metabolism and improve carbon efficiency. Additionally, metabolic modeling and 13C-metabolic flux analysis of mixed culture metabolism and cross-feeding offers a computational approach to complement experimental research for improved consortia performance.

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Plants are the source of various natural compounds with pharmaceutical and nutraceutical importance which have shown numerous health benefits with relatively fewer side effects. However, extraction of these compounds from native producers cannot meet the ever-increasing demands of the growing population due to, among other things, the limited production of the active compound(s). Their production depends upon the metabolic demands of the plant and is also subjected to environmental conditions, abundance of crop species and seasonal variations. Moreover, their extraction from plants requires complex downstream processing and can also lead to the extinction of many useful plant varieties. Microbial engineering is one of the alternative approaches which can meet the global demand for natural products in an eco-friendly manner. Metabolic engineering of microbes or pathway reconstruction using synthetic biology tools and novel enzymes lead to the generation of a diversity of compounds (like flavonoids, stilbenes, anthocyanins etc.) and their natural and non-natural derivatives. Strain and pathway optimization, pathway regulation and tolerance engineering have produced microbial cell factories into which the metabolic pathway of plants can be introduced for the production of compounds of interest on an industrial scale in an economical and eco-friendly way. While microbial production of phytochemicals needs to further increase product titer if it is ever to become a commercial success. The present review covers the advancements made for the improvement of microbial cell factories in order to increase the product titer of recombinant polyphenolic compounds.

Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti-Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (Histag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (10 8 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst−linker−affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 10 8 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.

Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology. Anthocyanins, the water-soluble colored pigments found in terrestrial plants that are responsible for the red, blue and purple coloration of many flowers and fruits, are extensively used in food and cosmetics industry; however, their current supply heavily relies on complex extraction from plant-based materials. A promising alternative is their sustainable production in metabolically engineered microbes. Here, we review the recent progress on anthocyanin biosynthesis in engineered bacteria, with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes, engineering of transportation, regulation of UDP-glucose supply, as well as process optimization. These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.

BackgroundAnthocyanins such as cyanidin 3-O-glucoside (C3G) have wide applications in industry as food colorants. Their current production heavily relies on extraction from plant tissues. Development of a sustainable method to produce anthocyanins is of considerable interest for industrial use. Previously, E. coli-based microbial production of anthocyanins has been investigated extensively. However, safety concerns on E. coli call for the adoption of a safe production host. In the present study, a GRAS bacterium, Corynebacterium glutamicum, was introduced as the host strain to synthesize C3G. We adopted stepwise metabolic engineering strategies to improve the production titer of C3G.ResultsAnthocyanidin synthase (ANS) from Petunia hybrida and 3-O-glucosyltransferase (3GT) from Arabidopsis thaliana were coexpressed in C. glutamicum ATCC 13032 to drive the conversion from catechin to C3G. Optimized expression of ANS and 3GT improved the C3G titer by 1- to 15-fold. Further process optimization and improvement of UDP-glucose availability led to ~ 40 mg/L C3G production, representing a > 100-fold titer increase compared to production in the un-engineered, un-optimized starting strain.ConclusionsFor the first time, we successfully achieved the production of the specialty anthocyanin C3G from the comparatively inexpensive flavonoid precursor catechin in C. glutamicum. This study opens up more possibility of C. glutamicum as a host microbe for the biosynthesis of useful and value-added natural compounds.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0990-z) contains supplementary material, which is available to authorized users.

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.

Metabolic engineering of microbial strains for the production of flavonoids of industrial interest has attracted great attention due to its promising advantages over traditional extraction approaches, such as independence of plantation, facile downstream separation, and ease of process and quality control. However, most of the constructed microbial production systems suffer from low production titers, low yields and low productivities, restricting their commercial applications. One important reason of the inefficient production is that the expression conditions and the detailed functions of the flavonoid pathway enzymes are not well understood. In this review, we have collected the biochemical properties, structural details, and genetic information of the enzymes in the flavonoid biosynthetic pathway as a guide for the expression and analysis of these enzymes in microbial systems. Additionally, we have summarized the engineering approaches used in improving the performances of these enzymes in recombinant microorganisms. Major challenges and future directions on the flavonoid pathway are also discussed.