Maximum Titer Research Articles

Promoter engineering with programmable upstream activating sequences in Aspergillus Niger cell factory

BackgroundAspergillus niger is an important industrial filamentous fungus used to produce organic acids and enzymes. A wide dynamic range of promoters, particularly strong promoters, are required for fine-tuning the regulation of gene expression to balance metabolic flux and achieve the high yields of desired products. However, the limited understanding of promoter architectures and activities restricts the efficient transcription regulation of targets in strain engineering in A. niger.ResultsIn this study, we identified two functional upstream activation sequences (UAS) located upstream of the core promoters of highly expressed genes in A. niger. We constructed and characterized a synthetic promoter library by fusing the efficient UAS elements upstream of the strong constitute PgpdA promoter in A. niger. It demonstrated that the strength of synthetic promoters was fine-tuned with a wide range by tandem assembly of the UAS elements. Notably, the most potent promoter exhibited 5.4-fold higher activity than the strongest PgpdA promoter reported previously, significantly extending the range of strong promoters. Using citric acid production as a case study, we employed the synthetic promoter library to enhance citric acid efflux by regulating the cexA expression in A. niger. It showed a 1.6-2.3-fold increase in citric acid production compared to the parent strain, achieving a maximum titer of 145.3 g/L.ConclusionsThis study proved that the synthetic promoter library was a powerful toolkit for precise tuning of transcription in A. niger. It also underscores the potential of promoter engineering for gene regulation in strain improvement of fungal cell factories.

Recycling of acid-pretreatment hydrolyzates of rice straw for high bioethanol production by S. cerevisiae and P. stipitis fermentation

ABSTRACT The current study developed a strategy to recycle the acid-pretreated hydrolyzate for multiple pretreatments of rice straw to increase the soluble sugar content in the medium, thereby enhancing bioethanol conversion. This process recovered a maximum glucose concentration of 10.4 g/L and xylose: 44.5 g/L in the recycled acid-pretreated rice straw hydrolyzate obtained after the third pretreatment. This resulted in a 2.4-fold increase in glucose recovery and a 2.3-fold increase in xylose recovery compared to the initial pretreatment process. For both glucose and xylose conversion strategies, a co-culture of hexose fermenting S. cerevisiae and P. stipitis, capable of fermenting both pentose and hexoses was employed. The maximum bioethanol titer achieved from the co-culture of S. cerevisiae and P. stipitis was 11.29 g/L. However, applying a sequential culture approach of S. cerevisiae followed by P. stipitis with an intermediate heat inactivation at 50°C resulted in an improvement of bioethanol titer to 12.39 g/L with productivity of 0.258 g/L/h. This led to an improvement in 2.9-fold and 1.2-fold bioethanol titer in comparison to sole fermentation by S. cerevisiae and P. stipitis, respectively. The reuse of acid pretreated hydrolyzate for high recovery of soluble sugar and subsequent fermentation by sequential culture strategy can maximize lignocellulosic bioethanol production.

Canine adenovirus serotype 2 isolation and determination of its cultivation parameters

Adenovirus infection in dogs caused by canine adenovirus serotype 2 predominantly results in respiratory disease typically manifested by respiratory tract lesions. Infectious laryngotracheitis is the most often recorded in dogs in the central part of the Russian Federation and its incidence tends to increase. Therefore, preventive immunization against this disease remains important. Primarily, the virus strains currently important and circulating in the particular territory shall be used for vaccine production to induce long-term and strong immunity in animals. The study was aimed at isolation of canine adenovirus type 2 remaining stable during five or more passages from the biological samples collected from animals with adenovirus infection signs as well as at determination of its cultivation parameters. As a result, five virus isolates were recovered, one of the recovered virus isolates had optimal properties for its use for vaccine production. Comparative analysis of continuous Vero, MDCK (NBL-2 and NBL-9 line) cell cultures as well as primarily trypsinized cell cultures (baby dog kidney, baby dog spleen, baby cat kidney, baby cat spleen) for their susceptibility to the recovered virus showed that MDCK (NBL-2 line) was the most susceptible. The virus cultivation parameters in this cell culture was determined at the next step. The following optimal conditions under which the virus accumulated to the maximum titres were determined: cell culture monolayer age for inoculation – 48 hours, multiplicity of infection – 0.01 TCID50/cell, preliminary holding time – 60 min, temperature – (37.0 ± 0.5) °С, cultivation period – 120 hours.

Multienzyme Cascade Synthesis of Rare Sugars From Glycerol in Bacillus subtilis.

Rare sugars are valuable and unique monosaccharides extensively utilized in the food, cosmetics, and pharmaceutical industries. Considering the high purification costs and the complex processes of enzymatic synthesis, whole-cell conversion has emerged as a significantly important alternative. The Escherichia coli strain was initially used in whole-cell synthesis of rare sugars. However, its pathogenic nature poses limitations to its widespread applications. Consequently, there is an urgent need to explore biologically safe strains for the efficient production of rare sugars. In this study, the generally regarded as safe (GRAS) strain Bacillus subtilis was employed as the chassis cells to produce rare sugars via whole-cell conversion. Three genes encoding alditol oxidase (AldO), L-rhamnulose-1-phosphate aldolase (RhaD), and fructose-1-phosphatase (YqaB) involved in rare sugars biosynthesis were heterogeneously expressed in B. subtilis to convert the only substrate glycerol into rare sugars. To enhance the expression levels of the relevant enzymes in B. subtilis, different promoters for aldO, rhaD, and yqaB were investigated and optimized in this system. Under the optimized reaction conditions, the maximum total production titer was 16.96g/L of D-allulose and D-sorbose with a conversion yield of 33.9% from glycerol. Furthermore, the engineered strain produced 26.68g/L of D-allulose and D-sorbose through fed-batch for the whole-cell conversion, representing the highest titer from glycerol reported to date. This study demonstrated an efficient and cost-effective method for thesynthesis of rare sugars, providing a food-grade platform with the potential to meet the growing demand for rare sugars in industries.

Engineering an Escherichia coli strain for enhanced production of flavonoids derived from pinocembrin.

Flavonoids are a structurally diverse group of secondary metabolites, predominantly produced by plants, which include a range of compounds with pharmacological importance. Pinocembrin is a key branch point intermediate in the biosynthesis of a wide range of flavonoid subclasses. However, replicating the biosynthesis of these structurally diverse molecules in heterologous microbial cell factories has encountered challenges, in particular the modest pinocembrin titres achieved to date. In this study, we combined genome engineering and enzyme candidate screening to significantly enhance the production of pinocembrin and its derivatives, including chrysin, pinostrobin, pinobanksin, and galangin, in Escherichia coli. By implementing a combination of established strain engineering strategies aimed at enhancing the supply of the building blocks phenylalanine and malonyl-CoA, we constructed an E. coli chassis capable of accumulating 353 ± 19mg/L pinocembrin from glycerol, without the need for precursor supplementation or the fatty acid biosynthesis inhibitor cerulenin. This chassis was subsequently employed for the production of chrysin, pinostrobin, pinobanksin, and galangin. Through an enzyme candidate screening process involving eight type-1 and five type-2 flavone synthases (FNS), we identified Petroselinum crispum FNSI as the top candidate, producing 82 ± 5mg/L chrysin. Similarly, from a panel of five flavonoid 7-O-methyltransferases (7-OMT), we found pinocembrin 7-OMT from Eucalyptus nitida to yield 153 ± 10mg/L pinostrobin. To produce pinobanksin, we screened seven enzyme candidates exhibiting flavanone 3-hydroxylase (F3H) or F3H/flavonol synthase (FLS) activity, with the bifunctional F3H/FLS enzyme from Glycine max being the top performer, achieving a pinobanksin titre of 12.6 ± 1.8mg/L. Lastly, by utilising a combinatorial library of plasmids encoding G. max F3H and Citrus unshiu FLS, we obtained a maximum galangin titre of 18.2 ± 5.3mg/L. Through the integration of microbial chassis engineering and screening of enzyme candidates, we considerably increased the production levels of microbially synthesised pinocembrin, chrysin, pinostrobin, pinobanksin, and galangin. With the introduction of additional chassis modifications geared towards improving cofactor supply and regeneration, as well as alleviating potential toxic effects of intermediates and end products, we anticipate further enhancements in the yields of these pinocembrin derivatives, potentially enabling greater diversification in microbial hosts.

ARTP/NTG Compound Mutagenesis Improved the Spinosad Production and the Insecticidal Virulence of Saccharopolyspora Spinosa.

Spinosad is an efficient and broad-spectrum environmentally friendly biopesticide, but its low yield in wild-type Saccharopolyspora spinosa limits its further application. ARTP/NTG compound mutagenesis was used in this study to improve the spinosad titer of S. spinosa and obtain a high-yield mutant-NT24. Compared with the wild-type strain, the fermentation cycle of NT24 was shortened by 2 days and its maximum titer of spinosad reached 858.3 ± 27.7 mg/L, which is 5.12 times more than for the same-period titer of the wild-type strain. In addition, RT-qPCR, resequencing, and targeted metabolomics showed that the upregulation of the key differential genes accD6, fadD, sdhB, oadA, and gntZ caused increased metabolic flux in the tricarboxylic acid cycle and pentose phosphate pathway, suggesting that the accumulation of pyruvate and short-chain acyl-CoA was the primary cause of spinosad accumulation in NT24. This study demonstrates the effectiveness of ARTP mutagenesis in S. spinosa, and provides new insights for the mechanism of spinosad biosynthesis and metabolic engineering in S. spinosa.

Development of a starch-fermenting Zymomonas mobilis strain for bioethanol production

BackgroundBiorefinery using microorganisms to produce biofuels and value-added biochemicals derived from renewable biomass offers a promising alternative to meet our sustainable energy and environmental goals. The ethanologenic strain Zymomonas mobilis is considered as an excellent chassis for constructing microbial cell factories for diverse biochemicals due to its outstanding industrial characteristics in ethanol production, high specific productivity, and Generally Recognized as Safe (GRAS) status. Nonetheless, the restricted substrate range constrains its application.ResultsThe truncated ice nucleation protein InaK from Pseudomonas syringae was used as an autotransporter passenger, and α-amylase was fused to the C- terminal of InaK to equip the ethanol-producing bacterium with the capability to ferment renewable biomass. Western blot and flow cytometry analysis confirmed that the amylase was situated on the outer membrane. Whole-cell activity assays demonstrated that the amylase maintained its activity on the cell surface. The recombinant Z. mobilis facilitated the hydrolysis of starch into oligosaccharides and enabled the streamlining of simultaneous saccharification and fermentation (SSF) processes. In a 5% starch medium under SSF, recombinant strains containing Peno reached a maximum titer of 13.61 ± 0.12 g/L within 48 h. This represents an increase of 111.0% compared to the control strain's titer of titer of 6.45 ± 0.25 g/L.ConclusionsBy fusing the truncated ice nucleation protein InaK with α-amylase, we achieved efficient expression and surface display of the enzyme on Z. mobilis. This fusion protein exhibited remarkable enzymatic activity. Its presence enabled a cost-effective bioproduction process using starch as the sole carbon source, and it significantly reduced the required cycle time for SSF. This study not only provides an excellent Z. mobilis chassis for sustainable bioproduction from starch but also highlights the potential of Z. mobilis to function as an effective cellular factory for producing high-value products from renewable biomass.

Production of Oncolytic Measles Virus in Vero Cells: Impact of Culture Medium and Multiplicity of Infection.

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107-109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media-a serum-containing medium (SCM), a serum-free medium (SFM) and two chemically defined media (CDM)-affect MeV production. We infected Vero cells at MOIs of 0.02, 0.2 or 2 TCID50 cell-1 and the lowest MOI resulted in the largest number of infected cells towards the end of the production period. However, this did not equate to higher maximum MeV titers, which were similar for all the MOIs. The medium had a moderate effect, generating maximum titers of 0.89-2.17 × 106, 1.08-1.25 × 106 and 4.58-9.90 × 105 TCID50 mL-1 for the SCM, SFM and CDM, respectively. Infection at a low MOI often required longer process times to reach maximum yields. On the other hand, a high MOI requires a large amount of MeV stock. We would therefore recommend a mid-range MOI of 0.2 TCID50 cell-1 for MeV production. Our findings show that SCM, SFM and CDM are equally suitable for MeV production in terms of yield and process time. This will allow MeV production in serum-free conditions, addressing the safety risks and ethical concerns associated with the use of serum.

Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Improvement of succinate production from methane by combining rational engineering and laboratory evolution in Methylomonas sp. DH-1

Recently, methane has been considered a next-generation carbon feedstock due to its abundance and it is main component of shale gas and biogas. Methylomonas sp. DH-1 has been evaluated as a promising industrial bio-catalyst candidate. Succinate is considered one of the top building block chemicals in the agricultural, food, and pharmaceutical industries. In this study, succinate production by Methylomonas sp. DH-1 was improved by combining adaptive laboratory evolution (ALE) technology with genetic engineering in the chromosome of Methylomonas sp. DH-1, such as deletion of bypass pathway genes (succinate dehydrogenase and succinate semialdehyde dehydrogenase) or overexpression of genes related with succinate production (citrate synthase, pyruvate carboxylase and phosphoenolpyruvate carboxylase). Through ALE, the maximum consumption rate of substrate gases (methane and oxygen) and the duration maintaining high substrate gas consumption rates was enhanced compared to those of the parental strain. Based on the improved methane consumption, cell growth (OD600) increased more than twice, and the succinate titer increased by ~ 48% from 218 to 323 mg/L. To prevent unwanted succinate consumption, the succinate semialdehyde dehydrogenase gene was deleted from the genome. The first enzyme of TCA cycle (citrate synthase) was overexpressed. Pyruvate carboxylase and phosphoenolpyruvate carboxylase, which produce oxaloacetate, a substrate for citrate synthase, were also overproduced by a newly identified strong promoter. The new strong promoter was screened from RNA sequencing data. When these modifications were combined in one strain, the maximum titer (702 mg/L) was successfully improved by more than three times. This study demonstrates that successful enhancement of succinic acid production can be achieved in methanotrophs through additional genetic engineering following adaptive laboratory evolution.

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.

Insights into the Genome of a New Strain Serratia rubidaea XU1 Isolated from Radioactive Soil and its Prodigiosin Production and Antimicrobial Properties.

The genus Serratia is a typical red bacterium involved in prodigiosin synthesis. Here, we report the genome sequence of Serratia rubidaea XU1, which was isolated from radiation-contaminated soil in Xinjiang, China. The genome of XU1 is composed of 4,972,898 base pairs with a GC content of 59.25%. The genome sequence contains 4707 genes and encodes 4573 proteins, 79 tRNAs, and 17 rRNAs. The prodigiosin biosynthesis gene cluster was identified and analyzed, showing a sequence similarity of 85.55-96.02% with Serratia rubidaea. After optimizing the biosynthesis process, XU1 was able to achieve a maximum titer of 574 units/cell of prodigiosin at a pH of 7.5 and a temperature of 25°C for 36h. Glycerol at 20g/L and beef extract at 5g/L were used as the carbon and nitrogen sources, respectively. Prodigiosin extracted from XU1 demonstrated inhibition of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The availability of the sequenced genome of XU1 will be greatly beneficial and contribute to complementary studies on the biosynthetic mechanisms of prodigiosin.

Harnessing Bacillus subtilis QY5 PP784163 for Bioethanol Production from Potato Peel Waste and Nutrient Recovery for Animal Feed: Maximizing Resource Efficiency

The present work focuses on the utilization of potato peel waste for the production of bioethanol. In the present study, extensive screening was undertaken to isolate amylolytic and cellulolytic microbes using starchy biomass. After confirming the chemical composition of potato peel waste (PPW), several trials were performed to enhance the amylase and cellulase production from Bacillus subtilis to hydrolyze the PPW in submerged fermentation. Optimization of physical parameters was performed using both commercial and indigenous media from enzymatically hydrolyzed PPW. Different routes of various combinations were designed to enhance bioethanol production. The maximum ethanol titer of 0.50% and 0.41% was recorded in Route B and A, i.e., separate saccharification and ethanol fermentation and consolidated fermentation. Simultaneous saccharification and fermentation (SSF) also measured a good ethanol yield of 0.46%. The fermented residual cake was checked for nutritional components and showed a high content of protein and amino acids because of the addition of unicellular yeasts. This cake can be utilized as an animal feed supplement.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

Biochemical characterization of a flavonoid O-methyltransferase from Artemisia annua and its application in tamarixetin production

The O-position methylation of flavonoids is widely recognized as a key modification that substantially boosts their bioactivity and bioavailability, leading to enhanced absorption. The use of specific O-methyltransferases is frequently necessary for various substrate modifications. Here, a novel Artemisia annua O-methyltransferase (AaOMT) gene was cloned, heterologously expressed in Escherichia coli, and its biochemical properties against various flavonoids were characterized. Amino acid and phylogenic analyses indicated that AaOMT is a new flavonoid O-methyltransferase that belongs to the Class II O-methyltransferases family. It could specifically methylate most flavonoids at the 4′-OH position unless flavanones. The optimal temperature and pH for in vitro reactions were 35 °C and 8.0, respectively. Kinetic analysis revealed that AaOMT exhibits the highest affinity and relatively high catalytic efficiency towards quercetin. Consequently, recombinant strains harboring AaOMT were used to convert quercetin into tamarixetin in vivo. Additionally, different vectors with promoters and E. coli hosts were screened to enhance tamarixetin production. By optimizing cultivation conditions and scaling up fermentation capacity, we achieved a maximum tamarixetin titer of 706.9 mg/L (∼2.24 mM) finally. This study provides a novel, efficient, and regioselective 4′-O-methyltransferase (4′-OMT) and a strategy for the mass production of those rare and high-value 4′-methoxyflavonoids through metabolic engineering in vivo.

Efficient carbon flux allocation towards D-pantothenic acid production via growth-decoupled strategy in Escherichia coli

For industrial strain construction, rational allocation of carbon flux is of paramount importance especially for decoupling cell growth and chemical productions to get maximum titer, rate, yield (TRY), which become Gordian Knot. Here, a temperature-sensitive switch and genetic circuits was used for effectively decoupling cell growth from D-pantothenic acid (DPA) production, along with systematically metabolic engineering including blocking redundant pathways of pyruvate and enhancing DPA driving force. Afterwards, rapid biomass accumulation only happened during growth stage, and subsequent high-efficient DPA production was initiated with reducing fermentation temperature. Finally, 97.20 g/L DPA and 0.64 g/g glucose conversion rate were achieved in 5-liter fed-batch fermentation. These undisputedly represent a milestone for the biosynthesis of DPA. With using strategies for decoupling cell growth from chemical productions, it would serve as “Alexander’s sword” to cut Gordian Knot to get industrial chassis cells with excellent TRY for de novo biosynthesis of valuable chemicals.

Multilevel metabolic engineering for enhanced synthesis of S-adenosylmethionine by Bacillus amyloliquefaciens.

To enhance the de novo synthesis of SAM, the effects of several key genes on SAM synthesis were examined based on modular strategy, and the key genes were manipulated to obtain an engineered strain with high SAM production. In Bacillus amyloliquefaciens HSAM6, the deletion of argG gene to block aspartic acid branching degradation increased SAM titer to 254.78 ± 15.91 mg/L, up 18% from HSAM6. Subsequently, deleting the moaA gene to boost the supply of 5-methyltetrahydrofolate led to the stunted growth and the plummeting yield of SAM. Further improvement of strain growth by overexpression of the citA gene, while SAM synthesis was not significantly enhanced. Finally, the maximum SAM titer (452.89 ± 13.42mg/L) was obtained by overexpression SAM2 gene using the multicopy plasmid. The deletion of argG gene and the overexpression of SAM2 gene significantly improved SAM synthesis in B. amyloliquefaciens.

Employment of light-inducible promoter in genetically engineered cyanobacteria for photosynthetic isobutanol production with simulated diurnal sunlight and CO2

Cyanobacteria are oxygen-evolving prokaryotes that can be engineered for biofuel production from solar energy, CO2, and water. Isobutanol (IB) has the potential to serve as an alternative fuel and important chemical feedstock. The research involves engineering Synechocystis sp. PCC 6803, for photosynthetic isobutanol production via the 2-keto-acid pathway and their cultivation in lab-scale photobioreactors. This synthetic pathway involves the heterologous expression of two enzymes, α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd), under a strong light-inducible promotor, psbA2, known to show increased gene expression under high light. The use of psbA2 could be a valuable strategy for isobutanol production as economic scaling up demands the utilization of natural sunlight, which also provides very high light intensity at midday, facilitating increased production. The study reports isobutanol production from engineered strains containing both pathway genes and with only kivd. In shake flask studies, the highest isobutanol titre of 75 mg L−1 (12th day) was achieved from an engineered strain DM12 under optimized light intensity. DM12 was cultivated in a 2 L flat panel photobioreactor, resulting in a maximum isobutanol titre of 371.8 mg L−1 (10th day) with 2 % CO2 and 200 μmol photons m−2 s−1. Cultivation of DM12 in a photobioreactor under mimic diurnal sunlight demonstrated the highest productivity of 39 mg L−1 day−1 with the maximum titre of 308.5 mg L−1 (9th day). This work lays the foundation for sustainable, large-scale biobutanol production using solar energy.

Isolation and chemical immobilization of E. coli‐specific bacteriophage with NH2‐MIL‐101(Fe) MOF, a high photoluminescence rod‐shaped microcrystals for low‐level bacteria detection

As concern raised by the World Health Organization (WHO) of antibiotic‐resistant and bio‐defensive bacteria, a metal–organic framework (MOF) based optical biosensor came into consideration for precise, quick, and sensitive detection of Escherichia coli (E. coli) (ATCC 10799) using bacteriophage as a bio‐recognition element. In the present study, amine‐functionalized Fe‐based MOF, i.e., NH2‐MIL‐101(Fe), was synthesized by the solvothermal method (approx. 531–1106 nm in size and 20 mV zeta potentials by DLS) and further characterized by SEM, XRD, ATR‐FTIR, UV–VIS, and photoluminescent (PL) spectroscopy. The lytic bacteriophage was isolated from a sewage sample, purified, and concentrated using the ultra‐centrifugation method and achieved a high titer of 7.3 × 1012 PFU/ml. The concentration, stability, and accessible receptor binding domains (RBDs) of the biorecognition element for binding with their analytes play an important role in developing sensitive and specific biosensor systems. To fulfill the mentioned criteria, optimized glutaraldehyde concentration was estimated at 0.25%, at 30 °C for conjugating maximum bacteriophage titer of 8.6 × 105 PFU/ml for each 1 mg amine functionalized iron‐based MOF. The synthesized detection probe has shown excellent photoluminescence and antibacterial activity and achieved a detection limit of 652 CFU/ml over a bacterial detection concentration range from 5.78 × 101 to 5.78 × 106 CFU/ml for E. coli with 10–12 min of response time, high specificity, and long‐term stability even at room temperature. Therefore, it can be inferred that this MOF‐based strategy can be helpful in the specific and sensitive detection of various bacterial pathogens using bacteriophage as a bio‐recognition element.