Protein engineering for the synthetic cell factory: Recent advance and perspective.

Synthetic cell factory offers substantial advantages in economically efficient production of biofuels, chemicals, and pharmaceutical compounds. However, to create a high-performance synthetic cell factory, precise regulation of cellular material and energy flux is essential. In this context, protein components including enzymes, transcription factor-based biosensors and transporters play pivotal roles. Protein engineering is a technology for creating novel protein variants with desired properties and functions by modifying target sequence in natural. This review focuses on summarizing the latest advancements of protein engineering in optimizing various aspects of synthetic cell factory, including: enhancing enzyme activity to eliminate production bottlenecks, altering enzyme selectivity to steer metabolic pathways towards desired products, modifying enzyme promiscuity to explore innovative routes, and improving the efficiency of transporters. Furthermore, the utilization of protein engineering to modify protein-based biosensors accelerates evolutionary process and optimizes the regulation of metabolic pathways. The remaining challenges and future opportunities of this technology are also discussed.

Enzyme engineering for functional lipids synthesis: recent advance and perspective.

Functional lipids, primarily derived through the modification of natural lipids by various processes, are widely acknowledged for their potential to impart health benefits. In contrast to chemical methods for lipid modification, enzymatic catalysis offers distinct advantages, including high selectivity, mild operating conditions, and reduced byproduct formation. Nevertheless, enzymes face challenges in industrial applications, such as low activity, stability, and undesired selectivity. To address these challenges, protein engineering techniques have been implemented to enhance enzyme performance in functional lipid synthesis. This article aims to review recent advances in protein engineering, encompassing approaches from directed evolution to rational design, with the goal of improving the properties of lipid-modifying enzymes. Furthermore, the article explores the future prospects and challenges associated with enzyme-catalyzed functional lipid synthesis.

Improvement of the viability of Tetragenococcus halophilus under acidic stress by forming the biofilm cell structure based on RNA-Seq and iTRAQ analyses.

BACKGROUND: Tetragenococcus halophilus is a halophilic lactic acid bacterium (LAB) isolated from soya sauce moromi. During the production of these fermented foods, acid stress is an inevitable environmental stress. In our previous study, T. halophilus could form biofilms and the cells in the biofilms exhibited higher cell viability under multiple environmental stresses, including acid stress. RESULTS: In this study, the effect of preformed T. halophilus biofilms on cell survival, cellular structure, intracellular environment, and the expression of genes and proteins under acid stress was investigated. The result showed that acid stress with pH 4.30 for 1.5 h reduced the live T. halophilus cell count and caused cellular structure damage. However, T. halophilus biofilm cells exhibited greater cell survival under acid stress than the planktonic cells, and biofilm formation reduced the damage of acid stress to the cell membrane and cell wall. The biofilm cells maintained a higher level of H+ -ATPase activity and intracellular ammonia concentration after acid stress. The RNA-Seq and iTRAQ technologies revealed that the genes and proteins associated with ATP production, the uptake of trehalose and N-acetylmuramic acid, the assembly of H+ -ATPase, amino acid biosynthesis and metabolism, ammonia production, fatty acid biosynthesis, CoA biosynthesis, thiamine production, and acetoin biosynthesis might be responsible for the stronger acid tolerance of T. halophilus biofilm cells together. CONCLUSION: These findings further explained the mechanisms that allowed LAB biofilm cells to resist environmental stress. © 2023 Society of Chemical Industry.

Formation of biofilm changed the responses of Tetragenococcus halophilus to ethanol stress revealed by transcriptomic and proteomic analyses.

Biofilms were found to promote the survival of Tetragenococcus halophilus, a functional halophilic lactic acid bacterium in the production of high-salt fermented foods under various environmental stresses including ethanol stress. Here, a comprehensive exploration of the response of T.halophilus biofilms and planktonic cells to ethanol stress was performed. Biofilms showed an ability to reduce death and damage of cell membrane and wall under 12% ethanol stress The formation of biofilm changed the characteristic of Fourier transformed infrared spectroscopy (FT-IR). RNA-seq technology and iTRAQ technology revealed the differential expression of genes and proteins in biofilm and planktonic cells with or without ethanol treatment. The differentially expressed genes and proteins played positive roles in the biosynthesis of polysaccharides, proteins, and DNA, benefitting biofilm matrix production. The shelter provided by biofilms and the differential expression of genes and proteins involved in citrate formation, malate utilization, and the biosynthesis of tryptophan, fatty acid, lipoteichoic acid, and peptidoglycan might contribute to the stress tolerance of biofilm cells together. Results presented in this study may contribute to our understanding of biofilm formation by T. halophilus and the roles of bacterial biofilm in stress tolerance.

A combination of bioinformatics analysis and rational design strategies to enhance keratinase thermostability for efficient biodegradation of feathers.

Keratinase has shown great significance and application potentials in the biodegradation and recycle of keratin waste due to its unique and efficient hydrolysis ability. However, the inherent instability of the enzyme limits its practical utilization. Herein, we obtained a thermostability-enhanced keratinase based on a combination of bioinformatics analysis and rational design strategies for the efficient biodegradation of feathers. A systematical in silico analysis combined with filtering of virtual libraries derived a smart library for experimental validation. Synergistic mutations around the highly flexible loop, the calcium binding site and the non-consensus amino acids generated a dominant mutant which increased the optimal temperature of keratinase from 40 °C to 60 °C, and the half-life at 60 °C was increased from 17.3 min to 66.1 min. The mutant could achieve more than 66% biodegradation of 50 g/L feathers to high-valued keratin product with a major molecular weight of 36 kDa. Collectively, this work provided a promising keratinase variant with enhanced thermostability for efficient conversion of keratin wastes to valuable products. It also generated a general strategy to facilitate enzyme thermostability design which is more targeted and predictable.

Review of arginase as a promising biocatalyst: characteristics, preparation, applications and future challenges.

As a committed step in the urea cycle, arginase cleaves l-arginine to form l-ornithine and urea. l-Ornithine is essential to: cell proliferation, collagen formation and other physiological functions, while the urea cycle itself converts highly toxic ammonia to urea for excretion. Recently, arginase was exploited as an efficient catalyst for the environmentally friendly synthesis of l-ornithine, an abundant nonprotein amino acid that is widely employed as a food supplement and nutrition product. It was also proposed as an arginine-reducing agent in order to treat arginase deficiency and to be a means of depleting arginine to treat arginine auxotrophic tumors. Targeting arginase inhibitors of the arginase/ornithine pathway offers great promise as a therapy for: cardiovascular, central nervous system diseases and cancers with high arginase expression. In this review, recent advances in the characteristics, structure, catalytic mechanism and preparation of arginase were summarized, with a focus being placed on the biotechnical and medical applications of arginase. In particular, perspectives have been presented on the challenges and opportunities for the environmentally friendly utilization of arginase during l-ornithine production and in therapies.

Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation.

Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive.

Improving the Intensity of Integrated Expression for Microbial Production.

Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

Phospholipids (PLs) know-how: exploring and exploiting phospholipase D for its industrial dissemination.

Owing to their numerous nutritional and bioactive functions, phospholipids (PLs), which are major components of biological membranes in all living organisms, have been widely applied as nutraceuticals, food supplements, and cosmetic ingredients. To date, PLs are extracted solely from soybean or egg yolk, despite the diverse market demands and high cost, owing to a tedious and inefficient manufacturing process. A microbial-based manufacturing process, specifically phospholipase D (PLD)-based biocatalysis and biotransformation process for PLs, has the potential to address several challenges associated with the soybean- or egg yolk-based supply chain. However, poor enzyme properties and inefficient microbial expression systems for PLD limit their wide industrial dissemination. Therefore, sourcing new enzyme variants with improved properties and developing advanced PLD expression systems are important. In the present review, we systematically summarize recent achievements and trends in the discovery, their structural properties, catalytic mechanisms, expression strategies for enhancing PLD production, and its multiple applications in the context of PLs. This review is expected to assist researchers to understand current advances in this field and provide insights for further molecular engineering efforts toward PLD-mediated bioprocessing.

Versatile strategies for bioproduction of hyaluronic acid driven by synthetic biology.

Owing to its outstanding water-retention ability, viscoelasticity, biocompatibility and non-immunogenicity, Hyaluronic acid (HA), a natural linear polymer alternating linked by d-glucuronic acid and N-acetylglucosamine, has been widely employed in cosmetic, medical and clinical applications. With the development of synthetic biology and bioprocessing optimization, HA production via microbial fermentation is an economical and sustainable alternative over traditional animal extraction methods. Indeed, recently Streptococci and other recombinant systems for HA synthesis has received increasing interests due to its technical advantages. This review summarizes the production of HA by microorganisms and demonstrates its synthesis mechanism, focusing on the current status in various production systems, as well as common synthetic biology strategies include driving more carbon flux into HA biosynthesis and regulating the molecular weight (MW), and finally discusses the major challenges and prospects.

The tale of a versatile enzyme: Molecular insights into keratinase for its industrial dissemination.

Keratinases are unique among proteolytic enzymes for their ability to degrade recalcitrant insoluble proteins, and they are of critical importance in keratin waste management. Over the past few decades, researchers have focused on discovering keratinase producers, as well as producing and characterizing keratinases. The application potential of keratinases has been investigated in the feed, fertilizer, leathering, detergent, cosmetic, and medical industries. However, the commercial availability of keratinases is still limited due to poor productivity and properties, such as thermostability, storage stability and resistance to organic reagents. Advances in molecular biotechnology have provided powerful tools for enhancing the production and functional properties of keratinase. This critical review systematically summarizes the application potential of keratinase, and in particular certain newly discovered catalytic capabilities. Furthermore, we provide comprehensive insight into mechanistic and molecular aspects of keratinases including analysis of gene sequences and protein structures. In addition, development and current advances in protein engineering of keratinases are summarized and discussed, revealing that the engineering of protein domains such as signal peptides and pro-peptides has become an important strategy to increase production of keratinases. Finally, prospects for further development are also proposed, indicating that advanced protein engineering technologies will lead to improved and additional commercial keratinases for various industrial applications.

Improving the biocatalytic performance of co-immobilized cells harboring nitrilase via addition of silica and calcium carbonate.

To improve nicotinic acid (NA) yield and meet industrial application requirements of sodium alginate-polyvinyl alcohol (SA-PVA) immobilized cells of Pseudomonas putida mut-D3 harboring nitrilase, inorganic materials were added to the SA-PVA immobilized cells to improve mechanical strength and mass transfer performance. The concentrations of inorganic materials were optimized to be 2.0% silica and 0.6% CaCO3. The optimal pH and temperature for SA-PVA immobilized cells and composite immobilized cells were both 8.0 and 45 °C, respectively. The half-lives of composite immobilized cells were 271.48, 150.92, 92.92 and 33.12 h, which were 1.40-, 1.35-, 1.22- and 1.63-fold compared to SA-PVA immobilized cells, respectively. The storage stability of the composite immobilized cells was slightly increased. The composite immobilized cells could convert 14 batches of 3-cyanopyridine with feeding concentration of 250 mM and accumulate 418 g ·L-1 nicotinic acid, while the SA-PVA immobilized cells accumulated 346 g L-1 nicotinic acid.

Recombinant expression and molecular engineering of the keratinase from Brevibacillus parabrevis for dehairing performance.

Keratinase is capable of distinctive degradation of keratin, which provides an eco-friendly approach for keratin waste management towards sustainable development. In this study, the recombinant keratinase (KERBP) from Brevibacillus parabrevis was successfully expressed in Escherichia coli. The purified KERBP had the specific activity of 6005.3 U/mg. It showed remarkable tolerance to various surfactants and also no collagenolytic activity. However, the moderate thermal stability limited its further application. Thus, protein engineering was further adopted to improve its stability. The variants of T218S, S236C and N181D were constructed by site-directed mutagenesis and combinatorial mutagenesis. Compared with the wild type, the t1/2 at 60 °C for the variants T218S, S236C and N181D were 3.05-, 1.18- and 1-fold increase, respectively. Moreover, the double variants N181D-T218S and N181D-S236C significantly improved thermostability with 5.1 and 2.9 °C increase of T50, and prolonging t1/2 at 60 °C with 4.09 and 1.54-fold, respectively. And the catalytic efficiency of the T218S and N181D-T218S variants was also significantly improved. Furthermore, the keratinase displayed favorable ability to dehair wool from skin within 7 h, which showed potential in leather dehairing. Our work contributes to a further insight into the thermostability of keratinase and offers a promising alternative for industrial leather application.

Efficient keratinase expression via promoter engineering strategies for degradation of feather wastes.

Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l-cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory.

Engineered Reversal of Function in Glycolytic Yeast Promoters.

Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine.

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates L-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL L-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

Atmospheric and room temperature plasma (ARTP) mutagenesis enables xylitol over-production with yeast Candida tropicalis.

Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d-xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry.

Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase.

Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that pro-peptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production.

Baker's yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.

Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories.

Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l(-1) of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l(-1)) and fatty alcohols (1.5 g l(-1)), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.