Molecular modification and biotechnological applications of microbial aspartic proteases.

The growing preference for incorporating microbial aspartic proteases in industries is due to their high catalytic function and high degree of substrate selectivity. These properties, however, are attributable to molecular alterations in their structure and a variety of other characteristics. Molecular tools, functional genomics, and genome editing technologies coupled with other biotechnological approaches have aided in improving the potential of industrially important microbial proteases by addressing some of their major limitations, such as: low catalytic efficiency, low conversion rates, low thermostability, and less enzyme yield. However, the native folding within their full domain is dependent on a surrounding structure which challenges their functionality in substrate conversion, mainly due to their mutual interactions in the context of complex systems. Hence, manipulating their structure and controlling their expression systems could potentially produce enzymes with high selectivity and catalytic functions. The proteins produced by microbial aspartic proteases are industrially capable and far-reaching in regulating certain harmful distinctive industrial processes and the benefits of being eco-friendly. This review provides: an update on current trends and gaps in microbial protease biotechnology, exploring the relevant recombinant strategies and molecular technologies widely used in expression platforms for engineering microbial aspartic proteases, as well as their potential industrial and biotechnological applications.

Biointerface engineering through amalgamation of gene technology and site-specific growth factor conjugation for efficient osteodifferentiation.

The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules.

Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Insights into cyanobacterial alkane biosynthesis.

Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels. Though biological production of alkanes has been reported in various microorganisms, the reports citing photosynthetic cyanobacteria as natural producers have been the most consistent for the long-chain alkanes and alkenes (C15-C19). However, the production of alkane in cyanobacteria is low, leading to its extraction being uneconomical for commercial purposes. In order to make alkane production economically feasible from cyanobacteria, the titre and yield need to be increased by several orders of magnitude. In the recent past, efforts have been made to enhance alkane production, although with a little gain in yield, leaving space for much improvement. Genetic manipulation in cyanobacteria is considered challenging, but recent advancements in genetic engineering tools may assist in manipulating the genome in order to enhance alkane production. Further, advancement in a basic understanding of metabolic pathways and gene functioning will guide future research for harvesting the potential of these tiny photosynthetically efficient factories. In this review, our focus would be to highlight the current knowledge available on cyanobacterial alkane production, and the potential aspects of developing cyanobacterium as an economical source of biofuel. Further insights into different metabolic pathways and hosts explored so far, and possible challenges in scaling up the production of alkanes will also be discussed.

Temperature-controlled molecular weight of hyaluronic acid produced by engineered Bacillus subtilis.

To produce high-, medium- and low-molecular-weight hyaluronic acid (HA) at different temperatures using engineered Bacillus subtilis expressing hyaluronidase (HAase) from leech. By overexpressing the HAase gene hya in the HA-producing strain WmB using temperature-sensitive plasmid pKSV7, the engineered strain WmB-PYh produced HA with different molecular weights (8.61 kDa at 32 °C, 0.615 MDa at 42 °C, and 6.19 MDa at 47 °C). In this study, the molecular weight of HA was regulated by using leech HAase expressed from a temperature-sensitive plasmid. We thus obtained different molecular weight HAs by using a single bacterial strain at different culture temperatures.

Microbial engineering to produce fatty alcohols and alkanes.

Owing to their high energy density and composition, fatty acid-derived chemicals possess a wide range of applications such as biofuels, biomaterials, and other biochemical, and as a consequence, the global annual demand for products has surpassed 2 million tons. With the exhausting petroleum reservoirs and emerging environmental concerns on using petroleum feedstock, it has become indispensable to shift to a renewable-based industry. With the advancement in the field of synthetic biology and metabolic engineering, the use of microbes as factories for the production of fatty acid-derived chemicals is becoming a promising alternative approach for the production of these derivatives. Numerous metabolic approaches have been developed for conditioning the microbes to improve existing or develop new methodologies capable of efficient oleochemical production. However, there still exist several limitations that need to be addressed for the commercial viability of the microbial cell factory production. Though substantial advancement has been made toward successfully producing these fatty acids derived chemicals, a considerable amount of work needs to be done for improving the titers. In the present review, we aim to address the roadblocks impeding the heterologous production, the engineering pathway strategies implemented across the range of microbes in a detailed manner, and the commercial readiness of these molecules of immense application.

Precise promoter integration improves cellulose bioconversion and thermotolerance in Clostridium cellulolyticum.

Lignocellulose has been used for production of sustainable biofuels and value-added chemicals. However, the low-efficiency bioconversion of lignocellulose greatly contributes to a high production cost. Here, we employed CRISPR-Cas9 editing to improve cellulose degradation efficiency by editing a regulatory element of the cip-cel gene cluster in Clostridium cellulolyticum. Insertion of a synthetic promoter (P4) and an endogenous promoter (P2) in the mspI-deficient parental strain (Δ2866) created chromosomal integrants, P4-2866 and P2-2866, respectively. Both engineered strains increased the transcript abundance of downstream polycistronic genes and enhanced in vitro cellulolytic activities of isolated cellulosomes. A high cellulose load of 20 g/L suppressed cellulose degradation in the parental strain in the first 150 h fermentation; whereas P4-2866 and P2-2866 hydrolyzed 29% and 53% of the cellulose, respectively. Both engineered strains also demonstrated a greater growth rate and a higher cell biomass yield. Interestingly, the Δ2866 parental strain demonstrated better thermotolerance than the wildtype strain, and promoter insertion further enhanced thermotolerance. Similar improvements in cell growth and cellulose degradation were reproduced by promoter insertion in the wildtype strain and a lactate production-defective mutant (LM). P2 insertion in LM increased ethanol titer by 65%. Together, the editing of regulatory elements of catabolic gene clusters provides new perspectives on improving cellulose bioconversion in microbes.

Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas.

Optimization of intracellular biosynthesis process involving regulation of multiple gene expressions is dependent on the efficient and accurate expression of each expression unit independently. However, challenges of analyzing intermediate products seriously hinder the application of high throughput assays. This study aimed to develop an engineering approach for unsterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or (P3HB4HB) by recombinant Halomonas bluephagenesis (H. bluephagenesis) constructed via coupling the design of GFP-mediated transcriptional mapping and high-resolution control of gene expressions (HRCGE), which consists of two inducible systems with high- and low-dynamic ranges employed to search the exquisite transcription level of each expression module in the presence of γ-butyrolactone, the intermediate for 4-hydroxybutyrate (4HB) synthesis. It has been successful to generate a recombinant H. bluephagenesis, namely TD68-194, able to produce over 36 g/L P3HB4HB consisting of 16 mol% 4HB during a 7-L lab-scale fed-batch growth process, of which cell dry weight and PHA content reached up to 48.22 g/L and 74.67%, respectively, in 36 h cultivation. HRCGE has been found useful for metabolic pathway construction.

Incorporating comparative genomics into the design-test-learn cycle of microbial strain engineering.

Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies.

Microbial production of bi-functional molecules by diversification of the fatty acid pathway.

Fatty acids that are chemically functionalized at their ω-ends are rare in nature yet offer unique chemical and physical properties with wide ranging industrial applications as feedstocks for bio-based polymers, lubricants and surfactants. Two enzymatic determinants control this ω-group functionality, the availability of an appropriate acyl-CoA substrate for initiating fatty acid biosynthesis, and a fatty acid synthase (FAS) variant that can accommodate that substrate in the initial condensation reaction of the process. In Type II FAS, 3-ketoacyl-ACP synthase III (KASIII) catalyses this initial condensation reaction. We characterized KASIIIs from diverse bacterial sources, and identified variants with novel substrate specificities towards atypical acyl-CoA substrates, including 3-hydroxybutyryl-CoA. Using Alicyclobacillus acidocaldarius KASIII, we demonstrate the in vivo diversion of FAS to produce novel ω-1 hydroxy-branched fatty acids from glucose in two bioengineered microbial hosts. This study unveils the biocatalytic potential of KASIII for synthesizing diverse ω-functionalized fatty acids.

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

BACKGROUND: Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. RESULTS: Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. CONCLUSIONS: We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.

Towards consolidated bioprocessing of biomass and plastic substrates for semi-synthetic production of bio-poly(ethylene furanoate) (PEF) polymer using omics-guided construction of artificial microbial consortia.

Poly(ethylene furanoate) (PEF) plastic is a 100% renewable polyester that is currently being pursued for commercialization as the next-generation bio-based plastic. This is in line with growing demand for circular bioeconomy and new plastics economy that is aimed at minimizing plastic waste mismanagement and lowering carbon footprint of plastics. However, the current catalytic route for the synthesis of PEF is impeded with technical challenges including high cost of pretreatment and catalyst refurbishment. On the other hand, the semi-biosynthetic route of PEF plastic production is of increased biotechnological interest. In particular, the PEF monomers (Furan dicarboxylic acid and ethylene glycol) can be synthesized via microbial-based biorefinery and purified for subsequent catalyst-mediated polycondensation into PEF. Several bioengineering and bioprocessing issues such as efficient substrate utilization and pathway optimization need to be addressed prior to establishing industrial-scale production of the monomers. This review highlights current advances in semi-biosynthetic production of PEF monomers using consolidated waste biorefinery strategies, with an emphasis on the employment of omics-driven systems biology approaches in enzyme discovery and pathway construction. The roles of microbial protein transporters will be discussed, especially in terms of improving substrate uptake and utilization from lignocellulosic biomass, as well as from depolymerized plastic waste as potential bio-feedstock. The employment of artificial bioengineered microbial consortia will also be highlighted to provide streamlined systems and synthetic biology strategies for bio-based PEF monomer production using both plant biomass and plastic-derived substrates, which are important for circular and new plastics economy advances.

Advances in CRISPR-Cas systems for gut microbiome.

CRISPR-Cas technology has revolutionized microbiome research by enabling precise genetic manipulation of microbial communities. This review explores its diverse applications in gut microbiome studies, probiotic development, microbiome diagnostics, pathogen targeting, and microbial community engineering. Engineered bacteriophages and conjugative probiotics exemplify CRISPR-Cas's capability for targeted bacterial manipulation, offering promising strategies against antibiotic-resistant infections and other gut-related disorders. CRISPR-Cas systems also enhance probiotic efficacy by improving stress tolerance and colonization in the gastrointestinal tract. CRISPR-based techniques in diagnostics enable early intervention by enabling fast and sensitive pathogen identification. Furthermore, CRISPR-mediated gene editing allows tailored modification of microbial populations, mitigating risks associated with horizontal gene transfer and enhancing environmental and health outcomes. Despite its transformative potential, ethical and regulatory challenges loom large, demanding robust frameworks to guide its responsible application. This chapter highlights CRISPR-Cas's pivotal role in advancing microbiome research toward personalized medicine and microbial therapeutics while emphasizing the imperative of balanced ethical deliberations and comprehensive regulatory oversight.

Promoter Identification and Optimization for the Response of Lactobacillus plantarum WCFS1 to the Gram-Negative Pathogen-Associated Molecule N-3-Oxododecanoyl Homoserine Lactone.

Quorum sensing (QS) in bacteria has been well studied as a cellular communication phenomenon for decades. In recent years, such systems have been repurposed for the use of biosensors in both cellular and cell-free contexts as well as for inducible protein expression in nontraditional chassis organisms. Such biosensors are particularly intriguing when considering the association between the pathogenesis of some bacteria and their signaling intermediates. Considering this relationship and considering the recent demonstration of the species Lactobacillus plantarum WCFS1 as both a synthetic biology chassis and an organism capable of detecting a pathogen-associated QS molecule, we wanted to develop this organism as a QS sentinel. We used an approach combining techniques from both systems and synthetic biology to identify a number of native QS-response genes and to alter associated promoter activity to tune the output of L. plantarum cultures exposed to N-3-oxododecanoyl homoserine lactone. The resulting engineered QS sentinel reinforces the potential of modified lactic acid bacteria (LAB) for use in human-health-promoting applications and also demonstrates a simple rational workflow to engineer sentinel organisms to respond to any environmental or chemical stimuli.

Microbial synthesis of glycosaminoglycans and their oligosaccharides.

Compared with chemical synthesis and tissue extraction methods, microbial synthesis of glycosaminoglycans (GAGs) is attractive because of the advantages of eco-friendly processes, production safety, and sustainable development. However, boosting the efficiency of microbial cell factories, precisely regulating GAG molecular weights, and rationally controlling the sulfation degree of GAGs remain challenging. To address these issues, various strategies, including genetic, enzymatic, metabolic, and fermentation engineering, have been developed. In this review, we summarize the recent progress in the construction of efficient GAG-producing microbial cell factories, regulation of the molecular weight of GAGs, and modification of GAG chains. Moreover, future studies, remaining challenges, and potential solutions in this field are discussed.

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.

Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.

Microbial engineering strategies for synthetic microplastics clean up: A review on recent approaches.

Microplastics are the small fragments of the plastic molecules which find their applications in various routine products such as beauty products. Later, it was realized that it has several toxic effects on marine and terrestrial organisms. This review is an approach in understanding the microplastics, their origin, dispersal in the aquatic system, their biodegradation and factors affecting biodegradation. In addition, the paper discusses the major engineering approaches applied in microbial biotechnology. Specifically, it reviews microbial genetic engineering, such as PET-ase engineering, MHET-ase engineering, and immobilization approaches. Moreover, the major challenges associated with the plastic removal are presented by evaluating the recent reports available.

Israel and the global synthetic biology ecosystem.

The field of synthetic biology emerged a few decades ago, following some key works of researchers in the USA, Europe, and the Far East. It reached Israel through academia and a few years later it finally got the attention of industry, venture capitals, and government authorities, especially the Israeli Innovation Authority, hoping to encourage entrepreneurs to establish startups in this field. Here we provide an overview of the activity of the field of synthetic biology in Israel, including historical notes, current strategy, prospects and developments, and further insight that are relevant to any stakeholders in the synthetic biology field.

Biosynthesis of monoterpenoid and sesquiterpenoid as natural flavors and fragrances.

Terpenoids are a large class of plant-derived compounds, that constitute the main components of essential oils and are widely used as natural flavors and fragrances. The biosynthesis approach presents a promising alternative route in terpenoid production compared to plant extraction or chemical synthesis. In the past decade, the production of terpenoids using biotechnology has attracted broad attention from both academia and the industry. With the growing market of flavor and fragrance, the production of terpenoids directed by synthetic biology shows great potential in promoting future market prospects. Here, we reviewed the latest advances in terpenoid biosynthesis. The engineering strategies for biosynthetic terpenoids were systematically summarized from the enzyme, metabolic, and cellular dimensions. Additionally, we analyzed the key challenges from laboratory production to scalable production, such as key enzyme improvement, terpenoid toxicity, and volatility loss. To provide comprehensive technical guidance, we collected milestone examples of biosynthetic mono- and sesquiterpenoids, compared the current application status of chemical synthesis and biosynthesis in terpenoid production, and discussed the cost drivers based on the data of techno-economic assessment. It is expected to provide critical insights into developing translational research of terpenoid biomanufacturing.