Microbial Biosynthesis Research Articles

Valinophos Reveals a New Route in Microbial Phosphonate Biosynthesis That Is Broadly Conserved in Nature.

Phosphonate natural products are potent inhibitors of cellular metabolism with an established record of commercialization in medicine and biotechnology. Although genome mining has emerged as an accelerated method for the discovery of new phosphonates, a robust framework of their metabolism is needed to identify the pathways most likely to yield compounds with desired activities. Here we expand our understanding of these natural products by reporting the complete biosynthetic pathway for valinophos, a phosphonopeptide natural product containing the unusual (R)-2,3-dihydroxypropylphosphonate (DHPPA) scaffold. The pathway was defined by several enzymatic transformations and intermediates previously unknown to phosphonate natural products. A dedicated dehydrogenase served as a new phosphoenolpyruvate mutase coupling enzyme. Notably, its reduction of phosphonopyruvate to phosphonolactate defined a new early branchpoint in phosphonate biosynthesis. Functionally interconnected kinase and reductase enzymes catalyzed reactions reminiscent of glycolysis and arginine biosynthesis to produce a transient, but essential, phosphonolactaldehyde intermediate. We demonstrate esterification of l-valine onto DHPPA as a new biochemical activity for ATP-Grasp ligase enzymes. Unexpectedly, a second amino acid ligase then adjoined additional amino acids at the valinyl moiety to produce a suite of DHPPA-dipeptides. The genes for DHPPA biosynthesis were discovered among genomes of bacteria from wide-ranging habitats, suggesting a wealth of unknown compounds that may originate from this core pathway. Our findings establish new biosynthetic principles for natural products and provide definition to unexplored avenues for bioactive phosphonate genome mining.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23–31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

Polyhydroxybutyrate (PHB) production from crude glycerol by genetic engineering of Rhodotorula glutinis

Polyhydroxybutyrate (PHB) is a member of the Polyhydroxyalkanoates (PHAs) family, which is generally produced through microbial biosynthesis. The synthetic biology tools are applied to transform PHB genes (PhaA, PhaB and PhaC) into an oleaginous yeast (Rhodotorula glutinis) genome. Western blot analysis confirms the expression of PhaA, PhaB and PhaC in the candidate transformants. The strain of R. glutinis #100–29 was selected to be the best PHB production candidate according to the shaker results. As compared to glucose, crude glycerol was known to be a suitable carbon source for R. glutinis cultivation. The PHB concentration, yield and content obtained in a 5-L fermenter using 30 g/L of crude glycerol were 2.87 g/L, 95.53 mg/g and 62% respectively. To the best of our knowledge, these should be the highest numbers obtained in yeast for PHB production, which indicates that strain #100–29 could be a potential PHB producer from crude glycerol.

On maintenance and metabolisms in soil microbial communities

Biochemistry is an essential yet undervalued aspect of soil ecology, especially when analyzing soil C cycling. We assume, based on tradition, intuition or hope, that the complexity of biochemistry is confined to the microscopic world, and can be ignored when dealing with whole soil systems. This opinion paper draws attention to patterns caused by basic biochemical processes that permeate the world of ecosystem processes. From these patterns, we can estimate activities of the biochemical reactions of the central C metabolic network and gain insights into the ecophysiology of microbial biosynthesis, growth, and maintenance energy requirements: important components of Carbon Use Efficiency (CUE). We show that glucose is processed via the Embden-Meyerhof-Parnas glycolysis in one soil, but via Pentose Phosphate or Entner-Doudoroff pathways in two other soils. However, notwithstanding this metabolic diversity, glucose use efficiency is high and thus substrate use for maintenance energy and overflow respiration is low in these soils. These results contradict current dogma, based on four decades of debate in soil ecology, that the maintenance energy demand in soil communities is a quantitative important, although variable, component of soil community energy metabolism. We identify three main shortcomings in our current understanding of substrate use efficiency: 1) in numeric and conceptual models, we lack appreciation of the strategies that microbes employ to quickly reduce energy needs in response to starvation; 2) in order to understand variation in CUE, we need to improve our understanding of the processes of exudation (including all changes in allocation of C from the cell soluble to insoluble fraction and extracellular environment) and microbial turnover; and 3) whether tracer experiments can be used to measure the long-term substrate use efficiency of soil microbial communities depends critically on the ability and speed with which non-growing cells take up tracer substrates and metabolism activates and subsequently de-activates in response to starvation, as well as on how cellular activities scale to the community level. To move the field of soil ecology forward, future research must consider the details of microbial ecophysiology and develop new tools that enable direct measurement of microbial functioning in intact soils. We submit that 13C metabolic flux analysis is one of those new tools.

New holistic approach for the management of biofilm-associated infections by myco-metabolites.

Biofilm-associated infections have increased excessively over the recent years due to the increased population having impaired immune systems or as a result of certain medical conditions like transplantation, cancer, and any other chronic ailments. The abrupt enhancement of antibiotic resistance and enhanced utilization of biomedical devices offer new opportunities for microbial colonization leading to the development of microbial biofilms. Total eradication of recalcitrant microbial biofilms demands the adoption of a holistic approach and since the fungal metabolites enriched with bioactive compounds show efficacy in inhibiting the multiple factors behind biofilm formation, the anti-biofilm activities of fungal metabolites need to be appraised. Being effective in preventing various steps of biofilm formation, including inhibition of surface adhesion and cell-to-cell communication through quorum quenching, blocking of quorum sensing receptors, and enzymes involved in microbial cell wall biosynthesis, targeting the virulence factors and finally killing of biofilm bound individual cells; myco-metabolites are found effective as a potent holistic anti-biofilm agent. The wide spectrum of bioactive substances of fungi and their anti-biofilm activities against different pathogens and their multitarget characteristics are very promising in the field of treating biofilm infections.

Selective biosynthesis of retinol in S. cerevisiae

The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a β-carotene producer by introducing β-carotene 15,15ʹ-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production.Graphical

Laccases—Versatile Enzymes Used to Reduce Environmental Pollution

The accumulation of waste and toxic compounds has become increasingly harmful to the environment and human health. In this context, the use of laccases has become a focus of interest, due to the properties of these versatile enzymes: low substrate specificity, and water formation as a non-toxic end product. Thus, we begin our study with a general overview of the importance of laccase for the environment and industry, starting with the sources of laccases (plant, bacterial and fungal laccases), the structure and mechanism of laccases, microbial biosynthesis, and the immobilization of laccases. Then, we continue with an overview of agro-waste treatment by laccases wherein we observe the importance of laccases for the biodisponibilization of substrates and the biodegradation of agro-industrial byproducts; we then show some aspects regarding the degradation of xenobiotic compounds, dyes, and pharmaceutical products. The objective of this research is to emphasize and fully investigate the effects of laccase action on the decomposition of lignocellulosic materials and on the removal of harmful compounds from soil and water, in order to provide a sustainable solution to reducing environmental pollution.

An inventory of early branch points in microbial phosphonate biosynthesis.

Microbial phosphonate biosynthetic machinery has been identified in ~5 % of bacterial genomes and encodes natural products like fosfomycin as well as cell surface decorations. Almost all biological phosphonates originate from the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) catalysed by PEP mutase (Ppm), and PnPy is often converted to phosphonoacetaldehyde (PnAA) by PnPy decarboxylase (Ppd). Seven enzymes are known or likely to act on either PnPy or PnAA as early branch points en route to diverse biosynthetic outcomes, and these enzymes may be broadly classified into three reaction types: hydride transfer, aminotransfer, and carbon-carbon bond formation. However, the relative abundance of these branch points in microbial phosphonate biosynthesis is unknown. Also unknown is the proportion of ppm-containing gene neighbourhoods encoding new branch point enzymes and potentially novel phosphonates. In this study we computationally sorted 434 ppm-containing gene neighbourhoods based on these seven branch point enzymes. Unsurprisingly, the majority (56 %) of these pathways encode for production of the common naturally occurring compound 2-aminoethylphosphonate (AEP) or a hydroxylated derivative. The next most abundant genetically encoded intermediates were phosphonoalanine (PnAla, 9.2 %), 2-hydroxyethylphosphonate (HEP, 8.5 %), and phosphonoacetate (PnAc, 6 %). Significantly, about 13 % of the gene neighbourhoods could not be assigned to any of the seven branch points and may encode novel phosphonates. Sequence similarity network analysis revealed families of unusual gene neighbourhoods including possible production of phosphonoacrylate and phosphonofructose, the apparent biosynthetic use of the C-P lyase operon, and a virus-encoded phosphonate. Overall, these results highlight the utility of branch point inventories to identify novel gene neighbourhoods and guide future phosphonate discovery efforts.

Glycosyltransferases: Mining, engineering and applications in biosynthesis of glycosylated plant natural products

UDP-Glycosyltransferases (UGTs) catalyze the transfer of nucleotide-activated sugars to specific acceptors, among which the GT1 family enzymes are well-known for their function in biosynthesis of natural product glycosides. Elucidating GT function represents necessary step in metabolic engineering of aglycone glycosylation to produce drug leads, cosmetics, nutrients and sweeteners. In this review, we systematically summarize the phylogenetic distribution and catalytic diversity of plant GTs. We also discuss recent progress in the identification of novel GT candidates for synthesis of plant natural products (PNPs) using multi-omics technology and deep learning predicted models. We also highlight recent advances in rational design and directed evolution engineering strategies for new or improved GT functions. Finally, we cover recent breakthroughs in the application of GTs for microbial biosynthesis of some representative glycosylated PNPs, including flavonoid glycosides (fisetin 3-O-glycosides, astragalin, scutellarein 7-O-glucoside), terpenoid glycosides (rebaudioside A, ginsenosides) and polyketide glycosides (salidroside, polydatin).

Bio-fabrication of thermozyme-based nano-biosensors: their components and present scenario.

An amalgamation of microbiology, biocatalysis, recombinant molecular biology, and nanotechnology is crucial for groundbreaking innovation in developing nano-biomedicines and sensoristics. Enzyme-based nano-biosensor finds prospective applications in various sectors (environmental, pharmaceutical, food, biorefineries). These applications demand reliable catalytic efficiency and functionality of the enzyme under an extreme operational environment for a prolonged period. Over the last few years, bio-fabrication of nano-biosensors in conjunction with thermozymes from thermophilic microbes is being sought after as a viable design. Thermozymes are known for their robustness, are chemically resistant toward organic solvents, possess higher durability for constant use, catalytic ability, and stability at elevated temperatures. Additionally, several other attributes of thermozymes like substrate specificity, selectivity, and sensitivity make them desirable in developing a customized biosensor. In this review, crucial designing aspects of enzyme-based nano-biosensors like enzyme immobilization on an electrode surface, new materials derived from microbial sources (biopolymers based nanocomposites), improvisation measures for sensitivity, and selectivity have been addressed. It also covers microbial biosynthesis of nanomaterials used to develop sensoristic devices and its numerous applications such as wastewater treatment, biorefineries, and diagnostics. The knowledge will pave the way toward creating consistent eco-friendly, economically viable nanostructured-based technologies with broad applicability and exploitation for industrial use in the near future.

Rhizophere analysis of auxin producers harboring the phenylpyruvate decarboxylase pathway

The 3-indole acetic acid (IAA) produced by microorganisms in the rhizosphere modulates root growth and physiology, but the analysis of these microorganisms relies on the isolation of culturable IAA producers as molecular tools are lacking. Microbial biosynthesis of IAA may involve several pathways, like the phenylpyruvate decarboxylase pathway (using the ppdC gene) present in many phytobeneficial microorganisms. Here, we tested the hypothesis that PCR primers could be developed to quantify ppdC+ microorganisms and assess ppdC allele diversity in the rhizosphere and bulk soil. Effective ppdC primers were obtained and validated in silico and using individual ppdC+ strains. By qPCR, they enabled to evidence high abundance of ppdC+ microorganisms in different types of bulk and rhizospheric soils, up to 8 log of ppdC copies per g of soil. OTUs obtained with Illumina sequencing of bulk and rhizospheric soil samples clustered in numerous clades encompassing almost all the known ppdC diversity in soil environments, and pointing to soil type and plant species as factors potentially shaping ppdC allelic diversity. In conclusion, these new primers enabled the first culture-independent analysis of the ppdC+ functional group of IAA producers in environmental samples.

Almond Consumption for 8 Weeks Altered Host and Microbial Metabolism in Comparison to a Control Snack in Young Adults

Background and Aims Almond consumption can improve cardiometabolic (CM) health. However, the mechanisms underlying those benefits are not well characterized. This study explored the effects of consuming a snack of almonds vs. crackers for 8 weeks on changes in metabolomic profiles in young adults (clinicaltrials.gov ID: NCT03084003). Methods Participants (n = 73, age: 18–19 years, BMI: 18–41 kg/m2) were randomly assigned to consume either almonds (2 oz/d, n = 38) or an isocaloric control snack of graham crackers (325 kcal/d, n = 35) daily for 8 weeks. Blood samples were collected at baseline prior to and at 4 and 8 weeks after the intervention. Metabolite abundances in the serum were quantified by hydrophilic interaction chromatography quadrupole (Q) time-of-flight (TOF) mass spectrometry (MS/MS), gas chromatography (GC) TOF MS, CSH-ESI (electrospray) QTOF MS/MS, and targeted analyses for free PUFAs, total fatty acids, oxylipins and endocannabinoids. Linear mixed model analyses with baseline-adjustment were conducted, and those results were used for enrichment and network analyses. Microbial community pathway predictions from 16S rRNA sequencing of fecal samples was done using PICRUST2. Results Almond consumption enriched unsaturated triglycerides, unsaturated phosphatidylcholines, saturated and unsaturated lysophosphatidylcholines, tricarboxylic acids, and tocopherol clusters (p < 0.05). Targeted analyses reveal lower levels of omega-3 total fatty acids (TFAs) overall in the almond group compared to the cracker group (p < 0.05). Microbial amino acid biosynthesis, and amino sugar and nucleotide sugar metabolism pathways were also differentially enriched at the end of the intervention (p < 0.05). Conclusions The study demonstrates the differential effects of almonds on host tocopherol, lipid, and TCA cycle metabolism with potential changes in microbial metabolism, which may interact with host metabolism to facilitate the CM benefits.

Deep Shotgun metagenomic and 16S rRNA analysis revealed the microbial diversity of lactic acid bacteria in traditional fermented foods of eastern Hainan, China.

The eastern part of Hainan, China, has a flat terrain and a suitable climate with abundant sunshine and rain. This unique environment makes the tropical microbial resources of natural fermented food unique and rich. Therefore, we combined Shotgun metagenomic sequencing, 16S rRNA sequencing and pure culture technology to analyze the microbial diversity, microbiota composition, species differences and correlation of 30 traditional fermented food samples collected from Wenchang, Qionghai, Wanning and Lingshui in the eastern part of Hainan province, and isolated, identified and preserved the microorganisms in them. The results showed that the microbial community structure differs significantly between samples from different regions and between different substrates. The alpha diversity of microorganisms in traditional fermented foods in the Wanning area was higher than those of the other three areas. The beta diversity indicated that the microbiota structural difference between Wanning and Qionghai was smaller. This is consistent with the fact that the precipitation in the Wanning area is the highest and similar to that of Qionghai. The alpha diversity of microorganisms was the highest in fermented vegetables, followed by fermented grains, and the lowest in fermented seafood. Beta diversity showed that the microbiota composition of fermented grains and fermented vegetables is very similar, but that of fermented seafood is significantly different. The results of microbiota structural analysis showed that firmicutes and proteobacteria are the dominant bacterial phyla, and Lactobacillus plantarum and Lactobacillus fermentum are the dominant species in traditional fermented foods in eastern Hainan. Lactic acid bacteria are the dominant species in traditional fermented foods from the eastern Hainan region of China, regardless of the substrate used in fermentation. According to the microbial functional characteristics, the microbial metabolism and biosynthesis pathways in traditional fermented foods in Hainan tend to be active. In addition, combined with pure culture technology, we isolated, identified and preserved 342 lactic acid bacteria strains from traditional fermented food in eastern Hainan province. This study helped us understand the different characteristics of microbial communities in tropical southern China and supplement the Lactobacillus species resource pool in tropical southern China. Moreover, it provided new insights and directions for the development and utilization of fermented foods.

Chemistry and biosynthesis of bacterial polycyclic xanthone natural products.

Covering: up to the end of 2021Bacterial polycyclic xanthone natural products (BPXNPs) are a growing family of natural xanthones featuring a pentangular architecture with various modifications to the tricyclic xanthone chromophore. Their structural diversities and various activities have fueled biosynthetic and chemical synthetic studies. Moreover, their more potent activities than the clinically used drugs make them potential candidates for the treatment of diseases. Future unraveling of structure activity relationships (SARs) will provide new options for the (bio)-synthesis of drug analogues with higher activities. This review summarizes the isolation, structural elucidation and biological activities and more importantly, the recent strategies for the microbial biosynthesis and chemical synthesis of BPXNPs. Regarding their biosynthesis, we discuss the recent progress in enzymes that synthesize tricyclic xanthone, the protein candidates for structural moieties (methylene dioxygen bridge and nitrogen heterocycle), tailoring enzymes for methylation and halogenation. The chemical synthesis part summarizes the recent methodology for the division synthesis and coupling construction of achiral molecular skeletons. Ultimately, perspectives on the biosynthetic study of BPXNPs are discussed.

Phosphorus and Nitrogen Limitation as a Part of the Strategy to Stimulate Microbial Lipid Biosynthesis

Microbial lipids called a sustainable alternative to traditional vegetable oils invariably capture the attention of researchers. In this study, the effect of limiting inorganic phosphorus (KH2PO4) and nitrogen ((NH4)2SO4) sources in lipid-rich culture medium on the efficiency of cellular lipid biosynthesis by Y. lipolytica yeast has been investigated. In batch cultures, the carbon source was rapeseed waste post-frying oil (50 g/dm3). A significant relationship between the concentration of KH2PO4 and the amount of lipids accumulated has been revealed. In the shake-flask cultures, storage lipid yield was correlated with lower doses of phosphorus source in the medium. In bioreactor culture in mineral medium with (g/dm3) 3.0 KH2PO4 and 3.0 (NH4)2SO4, the cellular lipid yield was 47.5% (w/w). Simultaneous limitation of both phosphorus and nitrogen sources promoted lipid accumulation in cells, but at the same time created unfavorable conditions for biomass growth (0.78 gd.m./dm3). Increased phosphorus availability with limited cellular access to nitrogen resulted in higher biomass yields (7.45 gd.m./dm3) than phosphorus limitation in a nitrogen-rich medium (4.56 gd.m./dm3), with comparable lipid yields (30% and 32%). Regardless of the medium composition, the yeast preferentially accumulated oleic and linoleic acids as well as linolenic acid up to 8.89%. Further, it is crucial to determine the correlation between N/P molar ratios, biomass growth and efficient lipid accumulation. In particular, considering the contribution of phosphorus as a component of coenzymes in many metabolic pathways, including lipid biosynthesis and respiration processes, its importance as a factor in the cultivation of the oleaginous microorganisms was highlighted.

Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

Recent Progress in Microbial Biosynthesis by Coculture Engineering

Recent Progress in Microbial Biosynthesis by Coculture Engineering

A point mutant in the promoter of transglutaminase gene dramatically increased yield of microbial transglutaminase from Streptomyces mobaraensis TX1

Microbial transglutaminase (TGase) is widely used in food processing because of its ability to catalyze protein cross-linking. In this study, ultraviolet (UV) mutagenesis was used to create a mutant of Streptomyces mobaraensis TX1 with a high TGase yield, with a maximum yield of 37.51 ± 0.46 U/mL after 32 h of flask fermentation, and a 2.15-fold increase compared to the wild-type (WT) strain. The transcriptional level of the transglutaminase gene increased 50-fold in the mutant strain compared to the WT strain, and a point mutation was found in the promoter of transglutaminase gene. The evaluation results of the promoter function revealed that the high yield of microbial transglutaminase was primarily caused by the point mutation. Our results provided a significant information on the high-yield mechanism of microbial transglutaminase biosynthesis.

Microorganism-mediated algal biomass processing for clean products manufacturing: Current status, challenges and future outlook

The global demand for clean products obtained from biobased resources has increased significantly with the rapid growth of the world’s population. In this context, microbially-produced compounds are highly attractive for their safety, reliability, being environment friendly and sustainability. Nevertheless, the cost of the carbon sources required for such approaches accounts for greater than 60% of the total expenses, which further limits the scaling up of industries. In recent years, algae have been used in numerous industrial areas because of their rapid growth rate, easy cultivation, ubiquity and survival in harsh conditions. Over the past decade, notable advances have been observed in the extraction of high-value compounds from algae biomass (ABs). However, few studies have investigated ABs as green substrates for microbial conversion into value-added products. This review presents the potential of ABs as the substrates for microbial growth to produce industrially-important products, which sheds light on the importance of the symbiotic relationship between ABs and microbial species. Moreover, the successful algal-bacterial gene transformation paves the way for accommodating green technology advancements. With the escalated need for natural pigments, biosurfactants, natural plastics and biofuels, ABs have been new resources for microbial biosynthesis of these value-added products, resolving the problem of high carbon consumption. In this review, the fermentative routes, process conditions, and accessibility of sugars are discussed, together with the related metabolic pathways and involved genes. To conclude, the full potential of ABs needs to be explored to support microbial green factories, producing novel bioactive compounds to meet global needs.

Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need.

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.