Microbial Cell Factories

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge, while incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to 7 genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expressions of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/L, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased FAD biosynthesis, enhanced flux entering the TCA cycle and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories. Impact StatementIn this study, yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and successfully incorporating modules for large-scale disulfide bond formation. KmYACs were stably propagated in K. marxianus without compromising the normal growth of the host, irrespective of the selection of telomeres (either Tetrahymena or K. marxianus) and configuration (either linear or circular). KmYACs notably enhanced the expressions of various heterologous proteins, with further yield improvement by supplementing lysine or arginine in the medium. Our findings affirm KmYAC as a robust and versatile platform for transferring large-scale function modules, demonstrating immense potential for both industrial applications and fundamental research.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase, along with PMT1 or PMT2, two ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of Scj1p had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. ImportanceOur study showcases a newly developed high throughput screening technique to identify yeast mutant strains that exhibit an enhanced capacity for recombinant protein production. Using a genome-wide approach, we show that vesicle trafficking plays a crucial role in protein production, as the genes associated with this process are notably enriched in our screen. Furthermore, we demonstrate that a specific set of gene deletions, which were not previously recognized for their impact on recombinant laccase production, can be effectively manipulated in combination to increase the production of heterologous proteins. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Slow cell growth and low biomass yields are hurdles for gas fermentation by acetogens. Microbial electrosynthesis (MES) utilizes acetogens as biocatalysts to reduce CO2 and produce commodity chemicals using electricity. However, limited electron supply in a bioelectrochemical system (BES) aggravates the poor cell growth of acetogens resulting in low productivities. Formate is a soluble C1 feedstock that can be produced by CO2 reduction (1). Thus, assimilation of formate can unburden the amount of electrons required for MES. Acetobacterium woodii posseses multiple pyruvate formate lyase (PFL) genes that are upregulated during formatotrophic growth. In this study, Clostridium ljugdahlii was engineered to heterologously express a pfl geneset of A. woodii to increase the cell growth of C. ljungdahlii during microbial electrosynthesis. Different combinations of pfl A and pfl B from A. woodii were tested in C. ljungdahlii to find the best working combination under control of Pfdx-riboswitch expression system. Expression of pfl B1 and pflA of A. woodii showed higher maximum OD of C. ljugdahlii under H2:CO2 condition with supplementation of 80 mM sodium formate. More than 40 mM of sodium formate caused significantly longer lag phase but the lag phase could be shortened after adaptation in 80 mM of sodium formate. At the end, the engineered strain showed improved cell growth (ODmax 0.22 {+/-} 0.05) and acetate production (21.8 {+/-} 5.6 mM) during microbial electrosynthesis compared to the control strain (ODmax 0.10 {+/-}0.06 and 10.2 {+/-}2.5 mM acetate). These results will be useful for strain development for microbial electrosynthesis, as well as gas fermentation. HighlightsO_LIDifferent combinations of pyruvate formate lyases (pfl Bs) and pyruvate formate lyase acting enzymes (pfl As) from Acetobacterium woodii were tested to improve the cell growth of Clostridium ljungdahlii during gas fermentation and microbial electrosynthesis C_LIO_LIHeterologous expression of pfl B1 and pfl A using riboswitch expression system improved cell growth of C. ljungdahlii under H2:CO2 condition, even without inducer and formate supplementation. C_LIO_LIHigh concentraton of sodium formate caused longer lag phase, which was shortened by adaptation when pfl from A. woodii was heterologously expressed. C_LIO_LICell growth and acetate production of the engineered C. ljungdahlii strain improved during microbial electrosynthesis C_LI

Production of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production. After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 {+/-} 0.3 fold and 2.9 {+/-} 0.8 fold more secretory Tfu0937 {beta}-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 {degrees}C, 30 {degrees}C and 25 {degrees}C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 {+/-} 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications.

The thermophilic bacterium Rhodothermus marinus has mainly been studied for its thermostable enzymes. More recently, the potential of using the species as a cell factory and in biorefinery platforms has been explored, due to the elevated growth temperature, native production of compounds such as carotenoids and EPSs, the ability to grow on a wide range of carbon sources including polysaccharides, and available genetic tools. A comprehensive understanding of the metabolism of production organisms is crucial. Here, we report a genome-scale metabolic model of R. marinus DSM 4252T. Moreover, the genome of the genetically amenable R. marinus ISCaR-493 was sequenced and the analysis of the core genome indicated that the model could be used for both strains. Bioreactor growth data was obtained, used for constraining the model and the predicted and experimental growth rates were compared. The model correctly predicted the growth rates of both strains. During the reconstruction process, different aspects of the R. marinus metabolism were reviewed and subsequently, both cell densities and carotenoid production were investigated for strain ISCaR-493 under different growth conditions. Additionally, the dxs gene, which was not found in the R. marinus genomes, from Thermus thermophilus was cloned on a shuttle vector into strain ISCaR-493 resulting in a higher yield of carotenoids. ImportanceA biorefinery converting biomass into fuels and value-added chemicals is a sustainable alternative to fossil fuel-based chemical synthesis. Rhodothermus marinus is a bacterium that is potentially well suited for biorefineries. It possesses various enzymes that degrade biomass, such as macroalgae and parts of plants (e.g. starch and xylan) and grows at high temperatures (55-77{degrees}C) which is beneficial in biorefinery processes. In this study, we reviewed the metabolism of R. marinus and constructed a metabolic model. Such a model can be used to predict phenotypes, e.g. growth under different environmental and genetic conditions. We focused specifically on metabolic features that are of interest in biotechnology, including carotenoid pigments which are used in many different industries. We described cultivations of R. marinus and the resulting carotenoid production in different growth conditions, which aids in understanding how carotenoid yields can be increased in the bacterium.

Clostridium ljungdahlii is an acetogen used for syngas fermentation and capable of microbial electrosynthesis. While C. ljungdahlii has potential for industrial application because of its broad spectrum of metabolites and substrates, including CO2 and CO, the efficiency of extracellular electron uptake in a bioelectrochemical system is low. Therefore, C. ljungdahlii was evolutionary adapted on iron as sole electron source with the aim to improve extracellular electron uptake. Over 38 batches, 95% of the culture was replaced with fresh medium biweekly to retain iron-attached C. ljungdahlii, leading to improved acetate production rates with each cycle. Eight isolated strains were tested on fructose, H2 and CO2, and iron to screen evolved mutants with desired mutations. Compared to the wild-type, growth on fructose was similar and growth on H2 and CO2 was, surprisingly, worse, with only minor differences between isolates. The isolated mutants produced acetate at a rate of 0.14 {+/-} 0.01 mM/d on iron, while the wild-type strain produced 0.75 {+/-} 0.14 mM/d. Whole genome sequencing of isolated mutants revealed 16 mutations, of which seven mutations were found in all isolates. Mostly, membrane proteins were mutated. In a BES reactor, acetate production ceased after day 1. The optical density (OD) of isolate #8 stopped increasing after day 2, reaching 0.122 {+/-} 0.005, followed by the production of formate and ethanol. The wild-type strain continued to grow until day 4, reaching an OD of 0.177 {+/-} 0.003. These results may indicate that C. ljungdahlii slows down growth and produces ethanol as an energy reserve as an evolutionary strategy for survival in an electron-limited environment.

BackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producing Clostridium species represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation. ResultsIn this study, the methylglyoxal synthase gene (mgsA) from Clostridium beijerinckii NCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain of Clostridium beijerinckii NCIMB 8052 was engineered by cloning and expressing mgsA and methylglyoxal/glyoxal reductase (mgR) from Clostridium pasteurianum ATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains of C. beijerinckii NCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol--the precursor of 1,2-propanediol--were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtype C. beijerinckii NCIMB 8052, C. pasteurianum ATCC 6013 and Clostridium tyrobutyricum ATCC 25755 were challenged with methylglyoxal, C. beijerinckii produced [~]0.1 g/L (S)-(+)-1,2-Propanediol, while C. tyrobutyricum produced traces of lactate. C. pasteurianum produced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain of C. beijerinckii expressing fused MgsA and MgR from C. pasteurianum ATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA. ConclusionsRecombinant and native strains of C. beijerinckii, C. pasteurianum and C. tyrobutyricum studied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboring mgsA (both homologous and heterologous)-- whereas the strain expressing both mgsA and mgR from C. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol--suggests that C. beijerinckii might possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product of mgR from C. pasteurianum represents a promising metabolic engineering candidate towards increasing butanol production.

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

In Pichia pastoris (Komagataella phaffii), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self-induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF-C1 metabolism, and it cannot be converted into carbon dioxide in an fdh1{Delta} strain. Under non-repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF-C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for an fdh1{Delta} strain on sorbitol and a non-disrupted strain on sorbitol-methanol. Considering a P. pastoris fdh1{Delta} strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol-free processes in P. pastoris.

BackgroundThe yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. ResultsWe recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. ConclusionsOur work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

Industrial biotechnology comprises the manufacturing of bulk chemicals and high-value end-products from renewable feedstocks, thus it presents a valuable aspect in the present transition from traditional-resource demanding manufacturing to sustainable solutions. The non-conventional yeast Debaryomyces hansenii encompasses halotolerant characteristics that ensures its use in industrial applications, and hence, its industrial importance. For this purpose, a comprehensive and holistic understanding of its behaviour and response to abiotic stresses is essential. Through high-throughput screening methods, using advanced robotics and automation devices, the present study enlightens intraspecies behavioural characteristics of novel D. hansenii strains in response to sodium, as well as their ability to tolerate abiotic stress in semi-controlled micro-fermentations and spot-test studies. A significantly improved performance under those abiotic stresses was observed under the presence of 1M NaCl. Moreover, a positive and summative effect on growth was also found in pH 4 and high salt content. Our results align with previous findings suggesting the halophilic (and not just halotolerant) behaviour of D. hansenii, which is now extensive to all the D. hansenii strains included in this study. Strain-specific differential responses to the presence of sodium were also observed, with some strains exerting a more notable induction by the presence of salt than the standard strain (CBS767). Furthermore, our study provides indications of the use of D. hansenii in industrial bioprocesses based on lignocellulosic biomass and non-lignocellulosic feedstocks.

While investigating the conversion of cellulosic biomass to starch-like materials for industrial use, it was observed that the overexpression of native ADP-glucose pyrophosphorylase GlgC in Escherichia coli led to the formation of insoluble polysaccharide granules within the cytoplasm, occupying a large fraction of the cell volume, as well as causing an overall increase in cellular polysaccharide content. TEM microscopy revealed that the granules did not have the lamellar structure of starch, but rather an irregular, clustered structure. On starvation, cells overexpressing GlgC appeared unable to fully degrade their polysaccharide material and granules were still clearly visible in cultures after 8 days of starvation. Interestingly, the additional overexpression of the branching enzyme GlgB eliminated the production of granules and led to a further increase in cellular polysaccharides. GlgC is generally thought to be responsible for the rate-limiting step of glycogen synthesis. Our interpretation of these results is that excess GlgC activity may cause the elongation of glycogen chains to outpace the addition of side branches, allowing the chains of adjacent glycogen molecules to reach lengths at which they spontaneously intertwine, forming dense clusters that are largely inaccessible to the host. However, upon additional upregulation of the GlgB branching enzyme, the branching of the polysaccharide is able to keep speed with the synthesis of linear chains, eliminating the granule phenotype. This study suggests potential avenues for increasing bacterial polysaccharide production and recovery. ImportanceIn this work, the polysaccharide stores of Escherichia coli were altered through the addition of extra copies of the bacterias own polysaccharide synthesis genes. In this way, bacteria were created that produced over twice the level of storage polysaccharide as a control strain, in the form of a granule that could potentially facilitate easy harvest. Another form of mutant Escherichia coli was created that produced over seven times the normal level of storage polysaccharide, and also grew to higher cell densities in liquid culture. In addition to increasing our understanding of glycogen synthesis, it is proposed that similarly modified bacteria, grown on inexpensive waste materials, may be a useful source of starch-like polysaccharides for industrial or agricultural use. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field.

The bacterial genus Rhodococcus comprises organisms that perform an oleaginous behavior under certain growth conditions and the ratio of carbon and nitrogen availability. Thus, Rhodococcus spp have outstanding biotechnological features as microbial producers of biofuel precursors, which would be used instead of lipids from crops. It was postulated that lipid and glycogen metabolism in Rhodococci are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying the catalytic steps redirecting sugar moieties to temporal storage molecules, such as glycogen and trehalose. In this work, we analyzed two glycosyl-transferases GT4 from R. jostii, RjoGlgAb and RjoGlgAc, which were annotated as -glucan--1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were recombinantly produced in E. coli BL21 (DE3) cells, purified to near homogeneity, and kinetically characterized. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase (EC 2.4.1.21) activity. Besides, both enzymes were actives as maltose-1P synthases (GlgM, EC 2.4.1.342), although to a different extent. In this scenario, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar behavior regarding kinetic parameters and glucosyl-donor (ADP-glucose) preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen. Also, this rhodococcal enzyme used glucosamine-1P as a catalytically efficient aglycon. On the other hand, both activities exhibited by RjoGlgAb depicted similar kinetic efficiency and a preference for short-branched -1,4-glucans. Curiously, RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure to function relationships. Results presented herein constitute a milestone regarding polysaccharide biosynthesis in Actinobacteria, leading to (re)discovery of methyl-glucose lipo-polysaccharide metabolism in Rhodococci.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

BackgroundRecombinant protein production in Escherichia coli (E. coli) is a widely used system in both academic and industrial research owing to its low cost and wide availability of genetic tools. Despite its advantages, this system still struggles with soluble expression of recombinant proteins. To address this, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. However, traditional tags such as small ubiquitin-related modifier (SUMO) and Glutathione S-transferase (GST) can interfere with protein folding or require removal post-translation, which adds complexity and cost to production. To circumvent these issues, smaller solubility tags (<10 kDa) are preferred. This study specifically focuses on an 11-amino acid solubility-enhancing tag (NT11) derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. ResultsA comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we increased its activity and identified key residues for further development. Screening with the alanine mutant library consistently led to at least a two-fold improvement in protein yield for three different proteins. We also discovered that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing protein expression constructs. With these new insights, we have successfully doubled the recombinant protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and an originally low-yielding human epidermal growth factor (hEGF), in E. coli. ConclusionThe further characterisation and development of the NT11 tag have provided valuable insights into the optimization process for such small tags and expanded our understanding of its potential applications. The ability of NT11 tag to be positioned at different locations within the protein construct without compromising its effectiveness to enhance recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings have the potential to simplify and enhance the efficiency of recombinant protein production.