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Abstract Tryptophol (IET) is a metabolite derived from L-tryptophan that can be isolated from plants, bacteria, and fungi and has a wide range of biological activities in living systems. Despite the fact that IET biosynthesis pathways exist naturally in living organisms, industrial-scale production of IET and its derivatives is solely based on environmentally unfriendly chemical conversion. With diminishing petroleum reserves and a significant increase in global demand in all major commercial segments, it becomes essential to develop new technologies to produce chemicals from renewable resources and under mild conditions, such as microbial fermentation. Here we characterized and engineered the less-studied L-tryptophan pathway and IET biosynthesis in the baker’s yeast Saccharomyces cerevisiae, with the goal of investigating microbial fermentation as an alternative/green strategy to produce IET. In detail, we divided the aromatic amino acids (AAAs) metabolism related to IET synthesis into the shikimate pathway, the L-tryptophan pathway, the competing L-tyrosine/L-phenylalanine pathways, and the Ehrlich pathway based on a modular engineering concept. Through stepwise engineering of these modules, we obtained a yeast mutant capable of producing IET up to 1.04 g/L through fed-batch fermentation, a ~ 650-fold improvement over the wild-type strain. Besides, our engineering process also revealed many insights about the regulation of AAAs metabolism in S. cerevisiae. Finally, during our engineering process, we also discovered yeast mutants that accumulate anthranilate and L-tryptophan, both of which are precursors of various valuable secondary metabolites from fungi and plants. These strains could be developed to the chassis for natural product biosynthesis upon introducing heterologous pathways.

Abstract Industrial biotechnology heavily relies on the microbial conversion of carbohydrate substrates derived from sugar- or starch-rich crops. This dependency poses significant challenges in the face of a rising population and food scarcity. Consequently, exploring renewable, non-competing carbon sources for sustainable bioprocessing becomes increasingly important. Ethanol, a key C2 feedstock, presents a promising alternative, especially for producing acetyl-CoA derivatives. In this review, we offer an in-depth analysis of ethanol's potential as an alternative carbon source, summarizing its distinctive characteristics when utilized by microbes, microbial ethanol metabolism pathway, and microbial responses and tolerance mechanisms to ethanol stress. We provide an update on recent progress in ethanol-based biomanufacturing and ethanol biosynthesis, discuss current challenges, and outline potential research directions to guide future advancements in this field. The insights presented here could serve as valuable theoretical support for researchers and industry professionals seeking to harness ethanol's potential for the production of high-value products. Graphic Abstract

Abstract In the post-genomic era, the demand for faster and more efficient protein production has increased, both in public laboratories and industry. In addition, with the expansion of protein sequences in databases, the range of possible enzymes of interest for a given application is also increasing. Faced with peer competition, budgetary, and time constraints, companies and laboratories must find ways to develop a robust manufacturing process for recombinant protein production. In this review, we explore high-throughput technologies for recombinant protein expression and present a holistic high-throughput process development strategy that spans from genes to proteins. We discuss the challenges that come with this task, the limitations of previous studies, and future research directions.

The inactivated vaccine CoronaVac is one of the most widely used COVID-19 vaccines globally. However, the longitudinal evolution of the immune response induced by CoronaVac remains elusive compared with other vaccine platforms. Here, we recruited 88 healthy individuals who received 3 doses of CoronaVac vaccine. We longitudinally evaluated their polyclonal and antigen-specific CD4+ T cells and neutralizing antibody response after receiving each dose of vaccine for over 300 days. Both the second and third doses of vaccine induced robust spike-specific neutralizing antibodies, with a third vaccine further increasing the overall magnitude of antibody response and neutralization against Omicron sublineages B.1.1.529, BA.2, BA.4/BA.5, and BA.2.75.2. Spike-specific CD4+ T cells and circulating T follicular helper (cTfh) cells were markedly increased by the second and third dose of CoronaVac vaccine, accompanied by altered composition of functional cTfh cell subsets with distinct effector and memory potential. Additionally, cTfh cells were positively correlated with neutralizing antibody titers. Our results suggest that CoronaVac vaccine–induced spike-specific T cells are capable of supporting humoral immunity for long-term immune protection.

Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion, but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield. Here, we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer. First, we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture. The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter. Next, through transcriptional engineering that alters transcription factor binding sites (TFBSs) and the first transcribed sequence, the truncated promoter PA256 with a dramatically higher transcription level was generated. When producing the superfolder green fluorescent protein (sfGFP) under 1% ethanol conditions, PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36. This superior expression mode was further validated using two secreted proteins, camelid antibody fragment (VHH) and endoxylanase (XynA). Furthermore, utilizing CRISPRi technology, ethanol utilization blocking strains were created, and PA256 was shown to be impaired in the phosphotransacetylase (PTA) knockdown strains, indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation. Finally, this platform was applied to produce the “de novo design” protein NEO-2/15, and by introducing the N-propeptide of CspB, NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation. To the best of our knowledge, this is the first report of NEO-2/15 secretory overexpression.

Abstract Herpes zoster is caused by reactivation of the varicella zoster virus (VZV). Researching and developing a herpes zoster vaccine will help to decrease the incidence of herpes zoster. To increase the bioreactor productivity, a serum-free HEK293 cell perfusion process with adenovirus vector herpes zoster (rAd-HZ) vaccine production was developed efficiently using the design of experiment (DoE) method. First, serum-free media for HEK293 cells were screened in both batch and semi-perfusion culture modes. Then, three optimal media were employed in a medium mixture design to improve cell culture performance, and the 1:1 mixture of HEK293 medium and MCD293 medium (named HM293 medium) was identified as the optimal formulation. On the basis of the HM293 medium, the relationship of critical process parameters (CPPs), including the time of infection (TOI), multiplicity of infection (MOI), pH, and critical quality attributes (CQAs) (adenovirus titer (Titer), cell-specific virus yield (CSVY), adenovirus fold expansion (Fold)) of rAd-HZ production was investigated using the DoE approach. Furthermore, the robust setpoint and design space of these CPPs were explored. Finally, the rAd-HZ production process with parameters at a robust setpoint (TOI = 7.2 × 106 cells/mL, MOI = 3.7, and pH = 7.17) was successfully scaled-up to a 3-L bioreactor with an alternating tangential flow system, yielding an adenovirus titer of 3.0 × 1010 IFU/mL, a CSVY of 4167 IFU/cells, a Fold of 1117 at 2 days post infection (dpi). The DoE approach accelerated the development of a HEK293 serum-free medium and of a robust adenovirus production process.

Abstract Terpenoids form the most diversified class of natural products, which have gained application in the pharmaceutical, food, transportation, and fine and bulk chemical industries. Extraction from naturally occurring sources does not meet industrial demands, whereas chemical synthesis is often associated with poor enantio-selectivity, harsh working conditions, and environmental pollutions. Microbial cell factories come as a suitable replacement. However, designing efficient microbial platforms for isoprenoid synthesis is often a challenging task. This has to do with the cytotoxic effects of pathway intermediates and some end products, instability of expressed pathways, as well as high enzyme promiscuity. Also, the low enzymatic activity of some terpene synthases and prenyltransferases, and the lack of an efficient throughput system to screen improved high-performing strains are bottlenecks in strain development. Metabolic engineering and synthetic biology seek to overcome these issues through the provision of effective synthetic tools. This review sought to provide an in-depth description of novel strategies for improving cell factory performance. We focused on improving transcriptional and translational efficiencies through static and dynamic regulatory elements, enzyme engineering and high-throughput screening strategies, cellular function enhancement through chromosomal integration, metabolite tolerance, and modularization of pathways. Graphical Abstract

Abstract Background Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L. Conclusion In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Pulmonary arterial hypertension (PAH) is a serious disease with reduced systemic circulation and low bioavailability associated with conventional and dosed therapy, which inhaled drugs can avoid. A mean pulmonary artery pressure (mPAP) of ≥25 mmHg (1 mmHg=0.133 kPa) at rest or ≥30 mmHg during exercise and a pulmonary capillary pressure or left atrial pressure (PLA) of ≤15 mmHg can be diagnosed with PAH. Pulmonary hypertension is classified into primary PAH and secondary PAH according to the presence or absence of principles or risk factors. The main symptoms of pulmonary hypertension include dyspnoea, syncope, weakness, chest pain, and the presence of varying degrees of peripheral oedema. It is a highly pathogenic and life-threatening disease and can lead to delays in treatment if not diagnosed in time. In the past few years, the studies related to this progressed slowly, which brought great harm to patients with PAH. Reports showed that patients diagnosed with PAH should receive routine preventative care, such as pneumococcal and influenza vaccinations. Inhalation therapy is mainly used for the treatment of respiratory diseases and is of great interest due to the concentration of the drug in the airways and lung tissues. Therefore, the present situation of pulmonary hypertension and the characteristics of inhalation preparation were reviewed in this paper to provide some related cue for the treatment of pulmonary hypertension. In the future, it is necessary to develop more treatment methods for pulmonary hypertension.

Abstract Background Glycolate is an important α-hydroxy carboxylic acid widely used in industrial and consumer applications. The production of glycolate from glucose in Escherichia coli is generally carried out by glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. Glycolate accumulation was significantly affected by nitrogen sources and isocitrate dehydrogenase (ICDH), which influenced carbon flux distribution between the tricarboxylic acid (TCA) cycle and the glyoxylate shunt, however, the mechanism was unclear. Results Herein, we used RNA-Seq to explore the effects of nitrogen sources and ICDH knockout on glycolate production. The Mgly534 strain and the Mgly624 strain (with the ICDH deletion in Mgly534), displaying different phenotypes on organic nitrogen sources, were also adopted for the exploration. Though the growth of Mgly534 was improved on organic nitrogen sources, glycolate production decreased and acetate accumulated, while Mgly624 achieved a balance between cell growth and glycolate production, reaching 0.81 g glycolate/OD (2.6-fold higher than Mgly534). To further study Mgly624, the significant changed genes related to N-regulation, oxidative stress response and iron transport were analyzed. Glutamate and serine were found to increase the biomass and productivity respectively. Meanwhile, overexpressing the arginine transport gene argT accelerated the cell growth rate and increased the biomass. Further, the presence of Fe2+ also speeded up the cells growth and compensated for the lack of reducing equivalents. Conclusion Our studies identified that ICDH knockout strain was more suitable for glycolate production. RNA-Seq provided a better understanding of the ICDH knockout on cellular physiology and glycolate production.

Abstract Background In terms of protein production, the internal environment of the host influences the activity of expression elements, thus affecting the expression level of the target protein. Native expression elements from a specific strain always function well in the original host. In the present study, to enhance the endoxylanase (XynA) production level in Corynebacterium glutamicum CGMCC1.15647 with its native expression elements, approaches to reduce host expression obstacles and to promote expression were evaluated. Results We identified the signal peptide of CspB2 in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it along with its promoter for the production of endoxylanase (XynA) in this strain. The native cspB2 promoter and cspB2 signal peptide are superior to the well-used cspB1 promoter and cspA signal peptide for XynA expression in C. glutamicum CGMCC1.15647, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest XynA secretion efficiency level in deep 24-well plates level (2492.88 U/mL) was achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively. In fed-batch cultivation, the highest XynA accumulation (1.77 g/L) was achieved in the culture supernatant after 44 h of cultivation. Conclusion Adaptation between the expression elements and the host is crucial for XynA production in C. glutamicum CGMCC1.15647. Strategies including host optimization, chromosomal integration, and coexistence of plasmids were useful for efficient protein production in C. glutamicum.

It is widely accepted that features such as pI, length, molecular mass and amino acid (AA) sequence have a significant influence on protein solubility. Here, we mainly focused on AA composition and explored those that most affected the soluble expression level of human serum albumin (HSA) domain antibody (dAb). The soluble expression and sequence of 65 dAb variants were analysed using clustering and linear modelling. Certain AAs significantly affected the soluble expression level of dAb, with the specific AA combinations being (S, R, N, D, Q), (G, R, C, N, S) and (R, S, G); these combinations respectively affected the dAb expression level in the broth supernatant, the level in the pellet lysate and total soluble dAb. Among the 20 AAs, R displayed a negative influence on the soluble expression level, whereas G and S showed positive effects. A linear model was built to predict the soluble expression level from the sequence; this model had a prediction accuracy of 80 %. In summary, increasing the content of polar AAs, especially G and S, and decreasing the content of R, was helpful to improve the soluble expression level of HSA dAb.

Abstract Background Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

A chemiluminescent immunoassay for human serum osteocalcin, or bone Gla protein, was established using a double-antibody sandwich model. Examination of the hook effect revealed that it was significantly reduced, and no hook effect was observed at an osteocalcin concentration of 4000 ng/mL. The established method showed good analytical performance and thermal stability. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 3.22%–5.64%, the interassay CV was 4.42%–7.25%, and the recovery rate was 93.22%–107.99%. Cross-reactivity (CR) was not observed with bovine, rat, mouse, rabbit, or porcine samples. The developed method showed a good correlation with the N-MID product from Roche. In total, 1069 clinical patient samples were measured using the reagent kit developed in this study.

In this study, a chemiluminescence immunoassay (CLIA) for human serum 25-hydroxyvitamin D (25(OH)D) was established by a competition model. In serum, more than 99% of total circulating 25(OH)D binds to protein and less than 1% of 25(OH)D is in free form (Jassil et al., 2017). Before measuring concentration of 25(OH)D in serum, a releasing procedure should be conducted. A new reagent is used to release binding 25(OH)D to free form. Streptavidin (SA) was labeled to magnetic beads by a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) method. Biotinylated VD was used as a competitor of 25(OH)D in samples. Anti-VD antibody (aby) was labeled to horseradish peroxidase (HRP) by EDC to react with 25(OH)D and biotinylated-VD molecules. The pretreated samples or standards were added into the reaction tube with biotin-VD and anti-VD aby-HRP, free 25(OH)D in the sample competes with biotinylated VD for binding to anti-VD aby-HRP, an SA-labeled magnetic particle is added to isolate the signal-generating complex, and the signal is inversely proportional to the 25(OH)D concentration in the sample. The method established shows good thermostability and performance. The limitation of detection (LoD) is 1.43 ng/mL. The intra-assay coefficient of variation (CV) is 3.66%–6.56%, the interassay CV is 4.19%–7.01%, and the recovery rate is 93.22%–107.99%. Cross-reactivity (CR) was remarkably low with vitamin D2, vitamin D3, 1, 25-dihydroxyvitamin D3, and 1, 25-dihydroxyvitamin D2. At the same time, the cross-reaction values with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% and 100%, respectively. The developed method shows good correlation with the total VD product from Roche and DiaSorin. 1096 clinical patient samples were measured with developed reagent kit in this study. 7 types of disease were involved, and the concentration of 25(OH)D is less than 30 ng/mL in 94.98% of patients.

Folic acid (FA) is an important vitamin for human growth, especially for pregnant women. FA deficiency is associated with megaloblastic anemia, neural tube defects, cardiovascular diseases, irritability, diarrhea, and psychiatric disorders. Normally, FA molecules bind to folate-binding protein (FBP) in the serum as complex. Before quantify the FA concentration, a releasing procedure should be conducted. Alkaline condition and tris(2-carboxyethyl)phosphine (TCEP) are used to release binding FA to freeing state. In this work, a chemiluminescence immunoassay (CLIA) for human serum FA was established by competition model. Streptavidin (SA) was labeled to magnetic beads by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDAC/NHS) method. Activated biotin molecules were labeled to FBP molecules purified from milk. FA was labeled to horseradish peroxidase (HRP) by EDAC to activate the FA molecules. The pretreated samples or standards were added into the reaction tube with biotin-FBP and FA-horseradish peroxidase (HRP), FA in the sample compete with FA-HRP for binding to biotin-FBP, the signal is inversely proportional to the FA concentration. The method established shows good thermostability and performance. The limitation of detection (LOD) is 0.44 ng/mL. The intra-assay coefficient of variation (CV) is 3.6%–7.1%, the interassay CV is 4.2%–7.5%, and the recovery rate is 92.1%–103.5%. Cross reactivity (CR) was remarkably low with aminopterin, folinic acid, and methotrexate. The method shows good correlation with the FA CLIA product from Beckman Coulter; the equation is y = 0.9618x−0.1434 while the R2 value is 0.9224. The established method is sensitive, rapid, and accurate which can fully satisfy for the clinical requirement.

Corynebacterium glutamicum (C. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. Herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of C. glutamicum. Two C. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (EGFP), were cultured in parallel in 5-L bioreactors to study the change in metabolism in the process of EGFP expression. The results revealed that EGFP expression had great effects on the growth and metabolism of C. glutamicum and contributed to metabolism-like anaerobic conditions as follows: glycolysis was enhanced, the TCA cycle was shunted, and Glu, Val, Met, lactate and acetate were accumulated to produce sufficient ATP for EGFP production and transfer. Many differentially expressed genes related to ribosomal protein, transcriptional regulators, and energy metabolism were found to be expressed in the presence of EGFP, laying the foundation for identifying genomic loci to change the flow of the host cell metabolism to improve the ability of expressing foreign proteins in C. glutamicum.

Dissolved oxygen (DO) is an important factor in the fermentation process of Corynebacterium glutamicum, which is a widely used aerobic microbe in bio-industry. Herein, we described RNA-seq for C. glutamicum under different DO levels (50%, 30% and 0%) in 5 L bioreactors. Multivariate data analysis (MVDA) models were used to analyze the RNA-seq and metabolism data to investigate the global effect of DO on the transcriptional distinction of the substance and energy metabolism of C. glutamicum. The results showed that there were 39 and 236 differentially expressed genes (DEGs) under the 50% and 0% DO conditions, respectively, compared to the 30% DO condition. Key genes and pathways affected by DO were analyzed, and the result of the MVDA and RNA-seq revealed that different DO levels in the fermenter had large effects on the substance and energy metabolism and cellular redox balance of C. glutamicum. At low DO, the glycolysis pathway was up-regulated, and TCA was shunted by the up-regulation of the glyoxylate pathway and over-production of amino acids, including valine, cysteine and arginine. Due to the lack of electron-acceptor oxygen, 7 genes related to the electron transfer chain were changed, causing changes in the intracellular ATP content at 0% and 30% DO. The metabolic flux was changed to rebalance the cellular redox. This study applied deep sequencing to identify a wealth of genes and pathways that changed under different DO conditions and provided an overall comprehensive view of the metabolism of C. glutamicum. The results provide potential ways to improve the oxygen tolerance of C. glutamicum and to modify the metabolic flux for amino acid production and heterologous protein expression.

WDR5 is a core component of the human mixed lineage leukemia-2 complex, which plays central roles in ER positive tumour cells and is a major driver of androgen-dependent prostate cancer cell proliferation. Given the similarities between breast and prostate cancers, we explore the potential prognostic value of WDR5 gene expression on breast cancer survival. Our findings reveal that WDR5 over-expression is associated with poor breast cancer clinical outcome in three gene expression data sets and BreastMark. The eQTL analysis reveals 130 trans-eQTL SNPs whose genes mapped with statistical significance are significantly associated with patient survival. These genes together with WDR5 are enriched with "cellular development, gene expression, cell cycle" signallings. Knocking down WDR5 in MCF7 dramatically decreases cell viability, but does not alter tumour cell response to doxorubicin. Our study reveals the prognostic value of WDR5 expression in breast cancer which is under long-range regulation of genes involved in cell cycle, and anthracycline could be coupled with treatments targeting WDR5 once such a regimen is available.