Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade.However,due to complexity of cellular metabolism,the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering.Recently,cell-free protein synthesis system(CFPS)has been emerging as an enabling alternative to address challenges in biomanufacturing.This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits(biosensors)to speed up design-build-test(DBT)cycles of metabolic engineering and synthetic biology.

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields.However,making such modifications can take substantial time.As a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform.We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild type.Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

Silk fibroin as an additive for cell-free protein synthesis

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing,point-of-need manufacturing,and responsive materials.Meanwhile,silk fibroin from the silk worm,Bombyx mori,has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes.In this work,we explore the effects of silk fibroin as an additive in cell-free protein synthesis(CFPS)reactions.Impacts of silk fibroin on CFPS activity and stability after drying,as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed.We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%,which we attribute to macromolecular crowding effects.However,we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes.Further,the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture.Crosslinking attempts did not impact CFPS activity,but did yield localized protein aggregates rather than a hydrogel.We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

CO_(2)-elevated cell-free protein synthesis

Gases are the vital nutrition of all organisms as the precursor of metabolism pathways.As a potential biological process,protein synthesis is inevitably regulated by gas transport and utilization.However,the effect of carbon dioxide(CO_(2))present in many metabolic pathways on protein synthesis has not been studied well.In this work,carbon dioxide combined with oxygen was employed for cell-free protein synthesis(CFPS)in the tube-in-tube reactor with precise control of gas concentration.In this in vitro system,gases could directly affect the protein synthesis process without transmembrane transport.Varied concentrations of carbon dioxide(0-1%)and constant oxygen concentration(21%)were employed for CFPS to assess the effects.The cell-free reactions with 0.3%CO_(2) and 21%O_(2) showed the highest protein yields.The combined effect of CO_(2) and O_(2) also resulted in relatively high protein expression under high oxygen conditions(0.3%CO_(2) and 100%O_(2)).Moreover,metabolomics assays were performed to gain insight into metabolic changes,which showed that CO_(2) slightly improved energy metabolism and redox balance.In particular,the extra supplied CO_(2) activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity.The exploration of CO_(2) on protein synthesis could provide guiding implications for basic studies and biomanufacturing.

Streamlining the preparation of “endotoxin-free” ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase

An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.

Enhancing boll protein synthesis and carbohydrate conversion by the application of exogenous amino acids at the peak flowering stage increased the boll Bt toxin concentration and lint yield in cotton

In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments(i.e., CK, the untreated control;LA1, five amino acids;LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum(i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3%in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase(GPT)activity, glutamate oxaloacetate transaminase(GOT) activity, glucose content, fructose content and soluble acid invertase(SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering(DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.

Preliminary Isolation of Hormone Stimulating Muscle Protein Synthesis in Thoracic Ganglia from the Prawn, Macrobrachium rosenbergii

The results of the previous studies seem to show the crustaceans can secrete growth hormone,which regulate their growth.However,more evidences are needed to determine it.So the following research was conducted.The extract of thoracic ganglia (TGE) of adult Macrobrachium rosenbergii was applied to a Sephadex G-50 column,the resulting second peak was further isolated by HPLC,and 9 fractions were obtained.Among the fractions only the second fraction significantly increased the protein synthesis of the abdomen muscle of the juvenile M.rosenbergii that was injected TGE.The increasing effects were related to the protein concentration of the injecting fractions.The molecular weight of the substance in the second fraction isolated by HPLC was below 3.4?kDa.The results indicate that the thoracic ganglia of M.rosenbergii could secrete a peptide hormone,which stimulates the muscle protein synthesis of prawn,and the hormone might be the growth hormone of the prawn.

RESPONSES TO ETHYLENE AND PROTEIN SYNTHESIS OF CARNATION FLOWERS

The symptoms of petal response to ethylene, including petal in-rolling and increase in ion leakage through cell membrane, were completely inhibited by treatment of transcription inhibitor a-amanitin as well as protein synthesis inhibitor--cycloheximide. It was observed in Dianthus caryophyllus L. cv. White Sim that protein synthesis in flowers declined and the responsiveness to ethylene increased as the flowers became matured. However, when the flowers were pre-treated with aminooxyacetic acid (AOA), an inhibitor of ethylene synthesis, the observed responsiveness to ethylene in flowers declined with age. These results suggested that responsiveness to ethylene in flowers may be due to a declination in the capacity of protein synthesis in flowers.

Leucine Supplementation in a Chronically Protein-Restricted Diet Enhances Muscle Weight and Postprandial Protein Synthesis of Skeletal Muscle by Promoting the mTOR Pathway in Adult Rats

Low protein intake causes a decrease in protein deposition in most animal tissues. The purpose of this study was to investigate whether leucine supplementation would increase the synthesis rate of protein and muscle weight in adult rats, which chronically consume only 58.8% of their protein requirements. Thirty-six male Sprague-Dawley rats were assigned to one of three dietary treatments including a 20% casein diet （CON）, a 10% casein ＋ 0.44% alanine diet （R）, and a 10% casein ＋ 0.87% leucine diet （RL）. After a 10 d dietary treatment, plasma amino acid levels were measured after feeding, the gastrocnemius muscles and soleus muscles were harvested and weighed, and the fractional synthesis rate （FSR） and mammalian target of rapamycin （mTOR） signaling proteins in skeletal muscle were measured. Regarding the plasma amino acid level, the RL group had the highest concentration of leucine （P 〈 0.05） and the lowest concentration of isoleucine （P 〈 0.05） among the three groups, and the CON group had a lower concentration of valine （P 〈 0.05） than the R and RL groups. Compared with the R and RE groups, the CON group diet significantly increased （P 〈 0.05） feed intake, protein synthesis rate, and the phosphorylation of eukaryutic initiation factor 4E binding protein 1 （4E-BP1）, and decreased the weight of abdominal adipose. Compared with the R group, the RL group significantly increased in gastrocnemius muscle weight, protein synthesis rate, and phosphorylation of both ribosomal protein $6 kinase 1 （56K1） and 4E-BP1. In conclusion, when protein is chronically restricted in adult rat diets, leucine supplementation moderately improves body weight gain and increases muscle protein synthesis through mTOR activation,

Protein synthesis modulation as a therapeutic approach for amyotrophic lateral sclerosis and frontotemporal dementia

Protein synthesis is essential for cells to perform life metabolic processes.Pathological alterations of protein content can lead to particular diseases.Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding,accumulation,aggregation or mislocalization occur.Some of them(like the unfolded protein response)represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis(also known as proteostasis).This is even more important in neurons,as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age.Several neurodegenerative pathologies such as Alzheimer’s,Parkinson’s,and Huntington’s diseases,amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct,unbalanced protein overload.In amyotrophic lateral sclerosis and frontotemporal dementia,the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa(TDP-43).TDP-43 is an RNA binding protein that participates in RNA metabolism,among other functions.Dysregulation of TDP-43(e.g.aggregation and mislocalization)can dramatically affect neurons,and this has been linked to disease development.Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum.These variants can be causative of degeneration onset and progression.Most neurodegenerative diseases(including amyotrophic lateral sclerosis and frontotemporal dementia)have no cure at the moment;however,modulating translation has recently emerged as an attractive approach that can be performed at several steps(i.e.regulating activation of initiation and elongation factors,inhibiting unfolded protein response activation or inducing chaperone expression and activity).This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis.We strive to highlight the importance of research on drugs that,not only restore protein imbalance without compromising translational activity of cells,but are also as safe as possible for the patients.

Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

3-bromo-4,5-bis（2,3-dibromo-4,5-dihydroxybenzyl）-l,2-benzenediol （1） is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B （PTP1B）. Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-（2＇-bromo-6＇-（3＂,4＂-dimethoxybenzyl）- 3 ＇,4 ＇-dimethoxybenzyl）-4,5 -dimethoxybenzene （2）, 2,3-dibromo- 1 -（2 ＇-bromo-6＇-（2 ＂-bromo-4＂,5 ＂-dimethoxy- benzyl）-3＇,4＇-dimethoxybenzyl）-4,5-dimethoxybenzene （3）, 3,4-dibromo-5-（2＇-bromo-6＇-（2＂-bromo-4＂,5＂- dihydroxybenzyl）-3＇,4＇-dihydroxybenzyl）pyrocatechol （4） and 3,4-dibromo-5-（2＇-bromo-6＇-（3＂,4＂- dihydroxybenzyl）-3＇,4＇-dihydroxybenzyl）pyrocatechol （5）. PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

RNA and Protein Requirements for Eukaryotic Selenoprotein Synthesis

Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress foIlowed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase. However, the functions of this enzyme did not explain many of the other effects of selenium deficiency. The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient. The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications. Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis,and the features that distinguish this pathway from the corresponding prokaryotic pathway.Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion. Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique 'recoding' process

A study on liver mitochondria respiration and protein synthesis in cold adapted rats

Mitochondria were isolated from normal and cold adapted rat livers.The respiratory func-tion of mitochondria in rat livers,including ADP:O ratio(P/O)and the respiratory control ratio(RCR),was determined by oxygen electrode method,The protein synthesis in mitochondria wasstudied by observing the incorporation of[~3H]-Leucine into mitochondria.Polyacrylamide gelelectrophoresis was carried out to detect the changes of the inner membrane proteins.It was shownthat the P/O and RCR decreased in cold adapted rats in the 2nd and 4th weeks and returned tothe control level in the 6th week,the protein synthesis of mitochondria decreased significantly incold adapted rats in 1,2 and 4 weeks;the electrophoretic pattern of the inner membrane proteinsin mitochondria from cold adapted rat livers revealed some new bands.

Progress in Chemical Synthesis of Peptides and Proteins

For the proteins that cannot be expressed exactly by cell expression technology (e.g., proteins with multiple posttranslational modifications or toxic proteins), chemical synthesis is an important substitute. Given the limited peptide length offered by solid-phase peptide synthesis invented by Professor Merrifield, peptide ligation plays a key role in long peptide or protein synthesis by ligating two small peptides to a long one. Moreover, high-molecular-weight proteins must be synthesized using two or more peptide ligation steps, and sequential peptide ligation is such an efficient way. In this paper, we reviewed the development of chemical protein synthesis, including solid-phase peptide synthesis, chemical ligation, and sequential chemical ligation. © 2017, The Author(s).

ALTERATIONS IN THE PROTEIN SYNTHESIS AND CONTENTS OF RIBOSOME AND POLYSOME IN LIVEROF SELENIUM－DEFICIENT RATS

In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats.The results demonstrated that the amounts of ribosome and polysome as well as the ratio of polysome to ribosome in liver PM-supernatant from the Se-deficient rats were all remarkahly decreased.In the meantime,the rate of protein synthesis expressed as radioactivity or 3H-leucine incorporated into protein in the PM-supernatant system also decreased significantly.The results suggest that the decreases of ribosomes and proportion of ribosomal aggregates in PM-supernatant may be responsible for the decrease of the protein synthesis activity in liver of the Se-deficient animals.

Potential Genes for Regulation of Milk Protein Synthesis in Dairy Goat Mammary Gland

The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages： days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution （three animals per group）. Mammary tissue RNA was isolated for quantitative real- time RT-PCR of four casein genes alpha-s 1 casein （CSN 1S 1 ）, alpha-s2 casein （CSN 1 S2）, beta-casein （CSN2） and casein kappa （CSN3）, four whey protein genes lactoglobulin （LGB）, laetalbumin （LALBA）, laetofarrin （LTF）, and Whey acidic protein （WAP） and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor （PRLR）, AKT1, signal transducers and activators of transcription 5 （STAT5）, E74-Like Factor 5 （ELF5）, eukaryotic translation initiation factor 4E binding protein 1 （EIF4E-BP1）, S6kinase （S6K） and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.

REVERSAL OF THE DOUBLE-STRANDED-RNA-INDUCED INHIBITION OF PROTEIN SYNTHESIS BY A CATALYTICALLY INACTIVE MUTANT OF THE PROTEIN KINASE PKR

The interferon-inducible-stranded-RNA-depedet protein kinase PKR has been implicated in both the antiviral aand cell growth-regulatory effects of the interferons.Over-expression of the wild-type form of this protein inhibits cell proliferation,whereas over-expression of inactive mutant forms transforms cells to a tumouri-genie phenotype.It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase.We have investigated this possibility using the rabbbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor elF-2. Addition of a highly purified inactive PKR mutant,synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by the low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA of in the presence of another inhibitory protein kinase,the haem-controlled repressor.Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA.These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.

Expression Regulation of Starch and Storage Protein Synthesis Related Genes in Rice Grains

Starch and the storage proteins are the main nutritious substances in crop grains,and their composition and content in grains play a decisive role in the grain quality of rice and other staple food crops.This review has mainly summarized the new advances in the expression regulation of starch and storage protein synthesis related genes in rice grains.Moreover,the challenges of the starch and storage protein synthesis substances in rice genetic improvement were also discussed.This review will provide important information for genetic improvement of grain quality in rice and,potentially,other staple cereals.

The Effect of Human Recombinant Interferon Gamma(hrIFN-γ)on hCG Secretion of Trophoblast and Protein Synthesis of Decidual Tissue in Vitro

In this study the effect of human recombinant interferon gamma hrIFN-γ)on hCG secretion of human first trimester trophoblast and protein synthesis of decidual tissue was investigated in vitro.The results indicated that hrIFN-γat the doses of 250 U/ml medium and 2500 U/ml medium decreased hCG secretion of trophoblast obviously(P<0.05, P<0.01)in a dose dependent manner.The effect of hrIFN-γon protein synthesis at the doses of 10 U to 1,000 U/ml medium inhibited the 3H leucine incorporation obviously.The cpm values between control and experimental groups were significantly different(P<0. 05) in a dosedependent manner.Furthermore its inhibitory effect is in a dose-dependent manner and was neutralized by IFN-γantiserum.The IFN-a at the doses used did not find any effect on protein synthesis in decidual tissue.

Effect of Chronic Supplementation with L-Arginine on the Expression of Proteins that Regulate Muscle Protein Synthesis in Rats Trained in High-Intensity Exercise

Arginine is a conditionally essential amino acid that has been correlated with muscle protein synthesis. In order to investigate the effect of chronic supplementation of L-arginine on muscle protein synthesis via mTOR （mammalian target of rapamycin）, and contribute to the new scientific discussions on this amino acid in this context, adult male Wistar rats weighing about 200 g each were used, divided into four groups： TA （trained arginine）, SA （sedentary arginine）, CT （diet-control trained）, and CS （diet-control sedentary）. The diets were based on proposal A1N-93 （American Institute of Nutrition-1993）, in which one of them was enriched with 2% of arginine and the other with a mix of nonessential amino acids. Training of the animals consisted of sessions composed of four series of 10 jumps in a tank of water. Jumps were performed with a load of 50% of animals＇ body weight, five days a week for six weeks. Blood analyses done were insulin, glucose, amino acids, IGF-1 （insulin-like growth factor 1）, 1GFBP-3 （insulin-like growth factor-binding protein 3）, urea, and creatinine, as well as muscle and liver IGF-1. Molecular analyses were for IRS-1 （insulin receptor substrate 1）, PKB （protein kinase B）, also known as Akt, roTOR, 4E-BP1 （eukaryotic initiation factor 4E-binding protein 1） and p70S6K （p70 S6 kinase） by Western Blotting method. As a result, no statistically significant differences were found in the parameters evaluated except for creatinine, which was higher for the groups supplemented with arginine.