The structure-directing role of heterologous seeds in the synthesis of zeolite

Zeolites have been widely used as catalysts,ion-exchangers,and adsorbents in chemical industries,detergent industry,steel industry,glass industry,ceramic industry,medical and healthfield,and environmentalfield,and recently applied in energy storage.Seed-assisted synthesis is a very effective approach in promoting the crystallization of zeolites.In some cases,the target zeolite cannot be formed in the absence of seed zeolite.In homologous seed-assisted synthesis,the structure of the seed zeolite is the same to that of the target zeolite,while the structure of the seed zeolite is different to that of the target zeolite in the heterologous seed-assisted synthesis.In this review,we briefly summarized the heterologous seed-assisted syntheses of zeolites and analyzed the structure-directing effect of heterologous seeds and surveyed the“common composite building units(CBUs)hypothesis”and the“common secondary building units(SBUs)hypothesis”.However,both hypotheses cannot explain all observations on the heterologous seed-assisted syntheses.Finally,we proposed that the formation of the target zeolite does need nuclei with the structure of target zeolite and the formation of the nuclei of the target zeolite can be promoted by either the undissolved seed crystals with the same CBUs or SBUs to the target zeolite or by the facilitated appropriate distribution of the specific building units due to the presence of the heterologous seed that does not have any common CBUs and SBUs with the target zeolite.

Rational Engineering of Secondary Metabolic Pathways in a Heterologous Host to Enable the Biosynthesis of Hibarimicin Derivatives with Enhanced Anti-Melanomic Activity

A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

A Case Report: High-Grade Urothelial Carcinoma in the Renal Pelvis with Complete Kidney and Ureter Duplication Featuring Heterologous Differentiation

This report describes a considerably rare case of high-grade urothelial carcinoma of the renal pelvis and ureter,presenting with heterologous differentiation,in a patient with bilateral duplicated kidneys.A 73-year-old male experienced intermittent gross hematuria for 5 months,accompanied by lower back and abdominal pain.Ultrasound and computed tomography scans revealed bilateral renal and ureteral duplication with multiple tumors in the left renal pelvis.A total nephroterectomy and bladder cuff resection were performed on the left two nephrons.Multiple space-occupying lesions were identified in the left renal pelvis and ureter.Histopathological examination showed poorly differentiated and diverse tumor cells,manifesting as sarcomatoid carcinoma,papillary adenocarcinoma,and infiltrating high-grade urothelial carcinoma.The tumor infiltrated the subcutaneous fibrous connective tissue of the renal pelvis and the full thickness of the ureter.Given the rarity of recurrent renal urothelial carcinoma with heterogeneous differentiation,comprehensive imaging and pathological assessments are vital to delineate the nature of the lesion and the direction of tissue pathological heterologous differentiation.These evaluations guide early radical surgical interventions,improving survival rates.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.

Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris

The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use tot industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects： （1） increase recombinant cell concentrations in fermentor to high density during growth phase; （2） effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; （3） decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.

Heterologous Expression of Camellia sinensis Late Embryogenesis Abundant Protein Gene 1(CsLEA1)Confers Cold Stress Tolerance in Escherichia coli and Yeast

Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.

Enhanced Riboflavin Production by Expressing Heterologous Riboflavin Operon from B.cereus ATCC14579 in Bacillus subtilis

Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.

Heterologous Expression of Rat Testis GABA_A Receptor β3t Splicing Variant in CHO Cells

Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.

Screening Testosterone Residue in the Edible Tissues of Bovine with a Polyclonal Antibody Based Heterologous Immunoassay

Testosterone（17β-hydroxyandrost-4-en-3-one）is a19-carbon steroid hormone with potent androgenic properties.It maintains testicular function and develop secondary male sex characteristics.It also has strong anabolic effects,which initiates increased protein synthesis in muscle and bone[1].In the1950s,the recognition of the growth promoting properties of such a hormone

Plant as Bioreactor for Producing Heterologous Proteins

Using plants to produce heterologous proteins makes it very attractive due to the potentially low costs. Using this procedure it is possible to produce medicinal protein for clinical applications with the plants bioreactors increasing gradually. The paper proposes the five major systems of the plant bioreactor as well as their advantage and disadvantage and the development of each system. Focuses on the five major systems of the plant bioreactor to produce vaccines, antibodies and medical protein and the research achievement at the present stage and the research on my laboratory. The key technology research of plant bioreactor such as new genes, new biological components, new technologies and new research methods related with plant bioreactor offer a work foundation for a long-term development in future.

Evaluation of Carbon and Electron Flow in Lactobacillus brevis as a Potential Host for Heterologous 1-Butanol Biosynthesis

Heterofermentative lactic acid bacterium Lactobacillus brevis may be considered as a promising host for heterologous butanol synthesis because of tolerance to butanol and ability to ferment pentose and hexose sugars from wood hydrolysates that are cheap and renewable carbohydrate source. Carbon and electron flow was evaluated in two L. brevis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis. Conditions required for generation of acetyl-CoA and NADH which are necessary for butanol biosynthesis have been determined. Key enzymes controlling direction of metabolic fluxes in L. brevis in various redox conditions were defined. In anaerobic glucose fermentation, the carbon flow through acetyl-CoA is regulated by aldehyde dehydrogenase ALDH possessing low affinity to NADH and activity (KmNADH= 200 μM, Vmax= 0.03 U/mg of total cell protein). Aerobically, the NADH-oxidase NOX (KmNADH= 25 μM, Vmax = 1.7 U/mg) efficiently competes with ALDH for NADH that results in formation of acetate instead of acetyl-CoA. In general, external electron acceptors (oxygen, fructose) and pentoses decrease NADH availability for native ethanol and recombinant butanol enzymes and therefore reduce carbon flux through acetyl-CoA. Pyruvate metabolism was studied in order to reveal redirection possibilities of competitive carbon fluxes towards butanol synthesis. The study provides a basis for the rational development of L. brevis strains producing butanol from wood hydrolysate.

Heterologous expression of the maize transcription factor ZmbHLH36 enhances abiotic stress tolerance in Arabidopsis

Basic helix-loop-helix(bHLH)transcription factors are widely distributed in eukaryotes,and in plants,they regulate many biological processes,such as cell differentiation,development,metabolism,and stress responses.Few studies have focused on the roles of bHLH transcription factors in regulating growth,development,and stress responses in maize(Zea mays),even though such information would greatly benefit maize breeding programs.In this study,we cloned the maize transcription factor gene ZmbHLH36(Gene ID:100193615,GRMZM2G008691).ZmbHLH36 possesses conserved domains characteristic of the bHLH family.RT-qPCR analysis revealed that ZmbHLH36 was expressed at the highest level in maize roots and exhibited different expression patterns under various abiotic stress conditions.Transgenic Arabidopsis(Arabidopsis thaliana)plants heterologously expressing ZmbHLH36 had significantly longer roots than the corresponding non-transgenic plants under 0.1 and 0.15 mol L^(−1) NaCl treatment as well as 0.2 mol L^(−1) mannitol treatment.Phenotypic analysis of soil-grown plants under stress showed that transgenic Arabidopsis plants harboring ZmbHLH36 exhibited significantly enhanced drought tolerance and salt tolerance compared to the corresponding non-transgenic plants.Malondialdehyde contents were lower and peroxidase activity was higher in ZmbHLH36-expressing Arabidopsis plants than in the corresponding non-transgenic plants.ZmbHLH36 localized to the nucleus when expressed in maize protoplasts.This study provides a systematic analysis of the effects of ZmbHLH36 on root growth,development,and stress responses in transgenic Arabidopsis,laying a foundation for further analysis of its roles and molecular mechanisms in maize.

Combined strategies for improving the heterologous expression of a novel xylanase from Fusarium oxysporum Fo47 in Pichia pastoris

Xylanase,an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat,has been found to improve the organizational structure of dough and thus increase its volume.In our past work,one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P.pastoris.It has shown significant potential in improving the quality of whole wheat bread,making it become a candidate for development as a new flour improver.After optimization of expression elements and gene dose,the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P.pastoris.In addition,12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain,and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL.Nevertheless,combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone.To further evaluate the industrial potential,the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter.These engineering strategies improved the expression of xylanase FXYL by more than 104-fold,providing valuable insights for the cost-effective industrial application of FXYL in the baking field.

Identification of missing CYP450 enzymes involved in paclitaxel biosynthesis and heterologous reconstitution of baccatin III

Paclitaxel,a tetracyclic diterpenoid,has garnered attention for its potent anti-cancer properties and intricate molecular structure,making it a significant target for chemical synthesis and biosynthesis[1].However,its natural sources are extremely limited,as it is derived exclusively from the bark of endangered genus Taxus plants,which contain paclitaxel in very low concentrations(0.01%−0.05%)[2-3].Recent advances in synthetic biology present promising opportunities to enhance paclitaxel levels in Taxus cell cultures or to enable the reconstitution of its production in heterologous hosts,such as yeast or tobacco(Nicotiana benthamiana).

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and 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 seven 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 expression levels 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 flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid 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.

Heterologous expression facilitates the discovery and characterization of marine microbial natural products

Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals.Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals.However,discovering compounds with new skeletons from marine microbes remains challenging.In recent decades,multiple approaches have been de-veloped to discover novel marine microbial natural products,among which heterologous expression has proven to be an effective method.Facilitated by large DNA cloning and comparative metabolomic technologies,a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters(BGCs)in heterologous hosts.Heterologous expression is advantageous for character-izing gene functions and elucidating the biosynthetic mechanisms of natural products.This review provides an overview of recent progress in heterologous expression-guided discovery,biosynthetic mechanism elucidation,and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.

Lysohexaenetides A and B,linear lipopeptides from Lysobacter sp.DSM 3655 identified by heterologous expression in Streptomyces

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.

A novel heterologous receptor-binding domain dodecamer universal mRNA vaccine against SARS-CoV-2 variants

There are currently approximately 4000 mutations in the SARS-CoV-2 S protein gene and emerging SARS-CoV-2 variants continue to spread rapidly worldwide.Universal vaccines with high efficacy and safety urgently need to be developed to prevent SARS-CoV-2 variants pandemic.Here,we described a novel self-assembling universal mRNA vaccine containing a heterologous receptorbinding domain(HRBD)-based dodecamer(HRBD^(dodecamer))against SARS-CoV-2 variants,including Alpha(B.1.1.7),Beta(B.1.351),Gamma(B.1.1.28.1),Delta(B.1.617.2)and Omicron(B.1.1.529).HRBD containing four heterologous RBD(Delta,Beta,Gamma,and Wild-type)can form a stable dodecameric conformation under T4 trimerization tag(Flodon,FD).The HRBD^(dodecamer)-encoding mRNA was then encapsulated into the newly-constructed LNPs consisting of a novel ionizable lipid(4N4T).The obtained universal mRNA vaccine(4N4T-HRBD^(dodecamer))presented higher efficiency in mRNA transfection and expression than the approved ALC-0315 LNPs,initiating potent immune protection against the immune escape of SARS-CoV-2 caused by evolutionary mutation.These findings demonstrated the first evidence that structure-based antigen design and mRNA delivery carrier optimization may facilitate the development of effective universal mRNA vaccines to tackle SARS-CoV-2 variants pandemic.