Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation

Animal-derived protein production is one of the major traditional protein supply methods,which continues to face increasing challenges to satisfy global needs due to population growth,augmented individual protein consumption,and aggravated environmental pollution.Thus,ensuring a sustainable protein source is a considerable challenge.The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins,which is an important way to solve the protein supply problem.This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis.Moreover,using artificial bioengineered milk and artificial bioengineered eggs as examples,the progress of food protein supply transition based on synthetic biology has been systematically summarized.Additionally,the future of food synthetic biology as a potential source of protein has been also discussed.By strengthening and innovating the application of food synthetic biology technologies,including genetic engineering and high-throughput screening methods,the current limitations of artificial foods for protein synthesis and production should be addressed.Therefore,the development and industrial production of new food resources should be explored to ensure safe,high-quality,and sustainable global protein supply.

Metabolomics and gene expression levels reveal the positive effects of teaseed oil on lifespan and aging process in Caenorhabditis elegans

As an irreplaceable dietary constituent,lipids play a vital important role in health,but their effects on aging process and longevity are still not well known yet.In this paper,the metabolic profiling and gene expression levels of Caenorhabditis elegans were investigated to explore the effects of different edible oils on senescence and lifespan.The results showed that teaseed oil(TO)could prolong the life expectancy and slow down the aging process of C.elegans.Compared to the control group,the intake of lard oil(LO)and TO increased the expression levels of genes related to inhibition of protein aggregation(akt-1,daf-16,hsf-1,hsp-16.2)and lipid metabolism(daf-7,daf-1,mdt-15,lipl-4,fat-5,fat-6,fat-7),with a more significant alteration in TO group.Metabolomics revealed that palm oil can upregulated plenty of fatty acids(palmitic acid,stearic acid,tetracosanoic acid),together with some amino acids(tryptophan,L-aspartate,L-valine)and carbohydrate(D-glucose),while the trend was opposite in TO group.Besides,moderate-to-strong correlations were found between differential metabolites and changed genes.In general,this paper claimed that TO could prolong lifespan and slow down aging process via regulating the lipids,amino acids and carbohydrates metabolism.

Distinct co‑occurrence patterns and driving forces of abundant and rare bacterial communities in the multispecies solid‑state fermentation process of cereal vinegar

Multispecies solid-state fermentation is a traditional processing technique for the traditional Chinese food,such as cereal vinegar,Baijiu,etc.Generally,few abundant and many rare microbes were involved in such processes,and the necessity and roles of the latter are less studied.Here the co-occurrence patterns of abundant and rare bacterial community and abiotic factors infuencing their community assembly were investigated in acetic acid fermentation following starter inoculation,using Zhenjiang aromatic vinegar as a model system.Abundant taxa that contribute to the function of accumulating acid exhibited a ubiquitous distribution while the distribution of rare taxa along the fermentation process unraveled.The species composition of the rare taxa signifcantly altered,but abundant taxa were maintained after inoculation.Moreover,the diversity of rare taxa changed more signifcantly than that of abundant taxa.Both abundant and rare sub-communities,which were contributed more with species turnover than species richness,were demonstrated to be driven by pH,acetic acid,ammonium nitrogen,and ethanol.Stochastic processes regulated the assembly of both sub-communities,but more prominent on rare sub-communities.Co-occurrence network was more governed by rare sub-communities,and the co-variations between microbial communities were predominantly positive,implying that rare taxa played more important role in the fermentation stability and network robustness.Furthermore,seven network connectors were identifed,and three of them belonged to rare taxa.These microbes of diferent modules were enriched at particular phases of fermentation.These results demonstrate the ecological signifcance of rare bacteria and provide new insights into understanding the abiotic factors infuence microbial structure in traditional fermented foods.

Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis

L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in Escherichia coli to promote L-tryptophan production

In this study,phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas(EMP)pathway and pentose phosphate(PP)pathway in Escherichia coli,thus increasing the L-tryptophan production.Firstly,the effects of disrupting EMP pathway on L-tryptophan production were studied,and the results indicated that the strain with deletion of phosphofructokinase A(i.e.,E.coli JW-5ΔpfkA)produced 23.4±2.1 g/L of L-tryptophan production.However,deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth,especially deletion of glucosephosphate isomerase.Next,the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase(zwf)and 6-phosphogluconate dehydrogenase(gnd)and thus increasing the L-tryptophan production(i.e.,26.5±3.2 g/L vs.21.7±1.3 g/L)without obviously changing the cell growth(i.e.,0.41 h^(-1) vs.0.44 h^(-1))as compared with the original strain JW-5.Finally,the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated.It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd(i.e.,E.coli JW-5 zwf243 gnd361ΔpfkA)produced 31.9±2.7 g/L of L-tryptophan,which was 47.0%higher than that of strain JW-5.In addition,the glucose consumption rate of strain JW-5 zwf243 gnd361ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5.The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.

Identification of acetic-acid tolerance of Saccharomyces cerevisiae strains by microsatellite markers

The aim of this paper is to use the microsatellites to evaluate acid-tolerance in Saccharomyces(S.) cerevisiae. Microsatellites have been widely used as the molecular marker to classify and identify S. cerevisiae strains, analyze genetic relationships among strains, and reveal genetic diversity of S. cerevisiae populations. In this paper, 25 key microsatellites of S. cerevisiae from 44 industrial yeast strains are investigated in the medium withconcentration gradients of acetic acid. Based on the analysis of correlations between the key microsatellite loci repeat numbers and acid-tolerance of the strains, the allele size of 4P14 a and 10P13 is positively related to acid-tolerance(p ? 0.05), the allele size of AT-X, 4P1 a and 10P8 are significantly negatively related to acid-tolerance(p ? 0.01). The above results provide informations on the molecular biodiversity of S. cerevisiae strains and can be a theoretical guidance for molecular marker assisted selection.

Ball milling pretreatment of corn straw and Pediococcus acidilactici fermentation for non‑ruminant diet

A new strategy was proposed to improve the utilization rate of corn straw by making non-ruminant feed products.The corn straw was ball milled,and the crushed straw was subjected to solid-state fermentation.After ball milling,the particle size of corn straw decreased signifcantly from 4.85±0.07 cm to 10–100μm,the content of neutral detergent fber and acid detergent fber decreased by 1.3%and 3.5%,respectively,and the total soluble sugar content increased by 16.2 mg/g.At the same time,the straw treated by ball milling as substrate could promote the rapid growth of Pediococcus acidilactici R30,the increase of organic acid production and further improvement of other nutrients during solid-state fermentation.In conclusion,the fermented straw products after ball milling could signifcantly improve the performance of pig feed in terms of digestibility and nutritional value,and better meet the feed needs of non-ruminant animals while saving resources.

Brucea javanica oil inhibits proliferation of hepatocellular carcinoma cells and induces apoptosis via the PI3K/AKT pathway

Background:Brucea javanica oil(BJO),distributed primarily in Southeast Asia,has long been utilized as a therapeutic agent for treating malignancies.However,its anticancer mechanisms are not clearly understood.The objective of this study was to examine the mechanisms underlying its treatment of hepatocellular carcinoma cells.Methods:CCK8 assay was used to evaluate cell viability.Hoechst33342 staining and flow cytometry analyses were used to examine apoptosis.Mito-Tracker Red CMXRos kit was used to measure the membrane potential of mitochondria.ATP assay kit was used to evaluate ATP levels.Western blots were used to assess the presence of AKT,adenosine monophosphate-activated protein kinase,Caspase3,Caspase9,Bax,and Bcl-2.Results:BJO inhibited the proliferation of hepatocellular carcinoma cells HepG2 in a time-and dose-dependent manner.It induced apoptosis,with the percentage of cells treated with 50–150μg/mL BJO increasing from 8.01%to 28.02%in a concentration-dependent manner(P<0.05,when 50μg/mL of BJO group compared with the control group;P<0.001,when 100 or 150μg/mL of BJO group compared with the control group).After exposed to BJO,the expression of C-caspase3,C-caspase9 and Bax upregulated while that of Bcl-2 downregulated.BJO suppressed the PI3K/AKT pathway and promoted phosphorylation of adenosine monophosphate-activated protein kinase,while repressing the phosphorylation of mechanistic target of rapamycin.Compared with treatment by BJO alone,the PI3K/AKT agonist 740Y-P increased the survival rate of HepG2 cells(P<0.01)and attenuated the inhibitory effect of BJO on cell apoptosis(P<0.05).Conclusion:BJO is capable of inhibiting proliferation of HepG2 cells and inducing apoptosis via the PI3K/AKT pathway.

Human Action Recognition Based on Supervised Class-Specific Dictionary Learning with Deep Convolutional Neural Network Features

Human action recognition under complex environment is a challenging work.Recently,sparse representation has achieved excellent results of dealing with human action recognition problem under different conditions.The main idea of sparse representation classification is to construct a general classification scheme where the training samples of each class can be considered as the dictionary to express the query class,and the minimal reconstruction error indicates its corresponding class.However,how to learn a discriminative dictionary is still a difficult work.In this work,we make two contributions.First,we build a new and robust human action recognition framework by combining one modified sparse classification model and deep convolutional neural network(CNN)features.Secondly,we construct a novel classification model which consists of the representation-constrained term and the coefficients incoherence term.Experimental results on benchmark datasets show that our modified model can obtain competitive results in comparison to other state-of-the-art models.

Consolidated bioprocess of corn stover to polysaccharide using Chaetomium globosum CGMCC 6882

Polysaccharide produced from medicinal endophytic fungus not only has applications in foods,but also exhibits multiple biological activities.In this work,an endophytic fungus Chaetomium globosum CGMCC 6882 could use corn stover to produce a polysaccharide(GCP-SC)by consolidated bioprocess and the titer of GCP-SC reached 3.2 g/L.The transcriptional levels of genes related to cellulose degradation(cbh,cdh,glu and egl)in C.globosum CGMCC 6882 were 4.38,3.85,3.13,and 2.17 folds compared to the control group when corn stover was used as the sole carbon source.Moreover,GCP-SC showed a time-and dose-dependent manner of inhibitory effect on A549 cells and the inhibitory rate reached 93.3%.This work provides meaningful data on agricultural residues utilization and facilitation of future relative resource conversion studies.

An effective cytokine combination for ex vivo expansion of porcine muscle stem cells

Cultured meat technology is a novel and promising strategy for sustainable and effective meat production.Muscle stem cells are widely used as seed cell populations because of their ease of access and myogenic differentiation potential.However,muscle stem cells are difficult to continuously propagate ex vivo and are heavily dependent upon serum for survival and growth,which impedes their commercial use as a cultured meat source.Herein,we identified an effective four-cytokine combination to promote long-term proliferation of porcine muscle stem cells using a serial elimination screening approach,which was consisted of long chain human insulin growth factor-1,platelet derived growth factor BB,basic fibroblast growth factor,and epidermal growth factor.The expansion of muscle stem cells with the four-cytokine combination achieved a 6.31×107-fold increase in cell number after 28 days of culture with retained cell myogenic differentiation potential,and most importantly,reduced the amount of fetal bovine serum required for cell culture by at least 5%.Furthermore,the four-cytokine combination exerted the pro-proliferative function by activating PI3K/Akt/mTOR and MEK/ERK signaling pathways.This approach provides for a new means by which to industrialize the production of cultured meat.

Cell-free synthesis system-assisted pathway bottleneck diagnosis and engineering in Bacillus subtilis

Metabolic engineering is a key technology for cell factories construction by rewiring cellular resources to achieve efficient production of target chemicals.However,the existence of bottlenecks in synthetic pathway can seriously affect production efficiency,which is also one of the core issues for metabolic engineers to solve.Therefore,developing an approach for diagnosing potential metabolic bottlenecks in a faster and simpler manner is of great significance to accelerate cell factories construction.The cell-free reaction system based on cell lysates can transfer metabolic reactions from in vivo to in vitro,providing a flexible access to directly change protein and metabolite variables,thus provides a potential solution for rapid identification of bottlenecks.Here,bottleneck diagnosis of the N-acetylneuraminic acid(NeuAc)biosynthesis pathway in industrially important chassis microorganism Bacillus subtilis was performed using cell-free synthesis system.Specifically,a highly efficient B.subtilis cell-free system for NeuAc de novo synthesis was firstly constructed,which had a 305-fold NeuAc synthesis rate than that in vivo and enabled fast pathway dynamics analysis.Next,through the addition of all potential key intermediates in combination with substrate glucose respectively,it was found that insufficient phosphoenolpyruvate supply was one of the NeuAc pathway bottlenecks.Rational in vivo metabolic engineering of NeuAc-producing B.subtilis was further performed to eliminate the bottleneck.By down-regulating the expression level of pyruvate kinase throughout the growth phase or only in the stationary phase using inhibitory Nterminal coding sequences(NCSs)and growth-dependent regulatory NCSs respectively,the maximal NeuAc titer increased 2.0-fold.Our study provides a rapid method for bottleneck diagnosis,which may help to accelerate the cycle of design,build,test and learn cycle for metabolic engineering.

Enhanced production of L-sorbose by systematic engineering of dehydrogenases in Gluconobacter oxydans

L-Sorbose is an essential intermediate for the industrial production of vitamin C(L-ascorbic acid).However,the formation of fructose and some unknown by-products significantly reduces the conversion ratio of D-sorbitol to L-sorbose.This study aimed to identify the key D-sorbitol dehydrogenases in Gluconobacter oxydans WSH-003 by gene knockout.Then,a total of 38 dehydrogenases were knocked out in G.oxydans WSH-003,and 23 dehydrogenase-deficient strains could increase L-sorbose production.G.oxydans-30,wherein a pyrroloquinoline quinone-dependent glucose dehydrogenase was deleted,showed a significant reduction of a by-product with the extension of fermentation time.In addition,the highest conversion ratio of 99.60%was achieved in G.oxydans MD-16,in which 16 different types of dehydrogenases were inactivated consecutively.Finally,the gene vhb encoding hemoglobin was introduced into the strain.The titer of L-sorbose was 298.61 g/L in a 5-L bioreactor.The results showed that the systematic engineering of dehydrogenase could significantly enhance the production of L-sorbose.

Synthesis of symmetrical medium-and long-chain triacylglycerols rich in arachidonic acid at sn-2 position for infant formula

Medium-and long-chain triacylglycerols(MLCT)rich in arachidonic acid(ARA)at sn-2 position were synthesized by a two-step enzymatic method.Firstly,sn-2 monoacylglycerols(MAG)were synthesized at a temperature of 25℃ by enzymatic alcoholysis.The MAG with 69.42%ARA at sn-2 position were obtained by solvent extraction and low temperature solvent crystallization.Secondly,the MLCT rich in ARA at sn-2 position and capric acid(CA)at sn-1,3 positions were produced by enzymatic esterification.Under the optimal conditions(MAG:CA=1:3(mol/mol),0.05 MPa vacuum,8% Lipozyme RM IM,5 h,25℃),the content of triacylglycerol was up to 93.60%.The triacylglycerol in the form of C10:0-C20:4-C10:0(including isomers)was about 40.43%.The ARA contents in the total and sn-2 fatty acid composition of the final product were 32.35%and 51.12%,respectively.MLCT rich in ARA at sn-2 position were successfully produced and the product has the potential application for functional food and infant formula.

CcpA mutants infuence selective carbon source utilization by changing interactions with target genes in Bacillus licheniformis

The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources.This process is mainly governed by the global regulator catabolite control protein A(CcpA),which can recognize and bind to multiple target genes that are widely distributed in metabolic pathways.Although the DNA-binding domain of CcpA has been predicted,the infuence of key amino acids on target gene recognition and binding has yet to be uncovered.In this study,the impact of Lys31,Ile42 and Leu56 on in vitro protein–DNA interactions and in vivo carbon source selective utilization was investigated.The results showed that alanine substitution of Lys31 and Ile42,located within the 3rd helices of the DNAbinding domain,signifcantly weakened the binding strength between CcpA and target genes.These mutations also lead to alleviated repression of xylose utilization in the presence of glucose.On the other hand,the Leu56Arg mutant in the 4th helices exhibited enhanced binding afnity compared with that of the wild-type one.When this mutant was used to replace the native one in B.licheniformis cells,the selective utilization of glucose over xylose increased.This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins.This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.

解聚型魔芋葡甘露聚糖的制备以及生理活性研究的进展（英文）

魔芋葡甘露聚糖是从魔芋块茎中提取的一种高分子水溶性多糖。近些年研究表明,其解聚产物,除了具有高溶解性和低粘度等良好的理化性质外,还具有调节微生物菌群结构、抗氧化、免疫调节等多种生理活性。本文重点综述了解聚型葡甘露聚糖的制备方法以及菌群调节功能。除此之外,对其抗氧化、免疫调节功能以及安全性评价也进行了全面的总结,为解聚型葡甘露聚糖的研究与应用提供一定的依据与思路。

Fabrication of 3D biomimetic composite coating with broadband antireflection, superhydrophilicity, and double p-n heterojunctions

传统的单个材料与二维(2D ) biomimetic 蛾眼睛结构被它的 narrowband antireflection 和单个功能的能力限制。克服这些劣势，我们利用了湿蚀刻，热水的合成为制作结合了化学氧化一三维(3D ) 有第三的材料的 biomimetic 蛾眼睛涂层(polypyrrole nanoparticles， TiO 2 nanorods，和 Si micropyramids，即， PPy/TiO 2/Si-p) 。这涂层把反射率归结为 <4% 在与低水从 200 ～ 2,300 nm 和展出显著 superhydrophilicity 的波长联系 1.8

Toward fine-tuned metabolic networks in industrial microorganisms

There are numerous microorganisms in nature capable of synthesizing diverse useful compounds;however,these natural microorganisms are generally inefficient in the production of target products on an industrial scale,relative to either chemical synthesis or extraction methods.To achieve industrial production of useful compounds,these natural microorganisms must undergo a certain degree of mutation or effective fine-tuning strategies.This review describes how to achieve an ideal metabolic fine-tuned process,including static control strategies and dynamic control strategies.The static control strategies mainly focus on various matabolic engineering strategies,including protein engineering,upregulation/downregulation,and combinatrorial control of these metabolic engineering strategies,to enhance the flexibility of their application in fine-tuned metabolic metworks.Then,we focus on the dynamic control strategies for fine-tuned metabolic metworks.The design principles derived would guide us to construct microbial cell factories for various useful compounds.

Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus

2-keto-D-gluconic acid(2-KGA)is a key precursor for synthesising vitamin C and isovitamin C.However,phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens.Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids,and high utilisation of glucose.In this study,the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-D-gluconic acid(5-KGA)and D-gluconic acid(D-GA)in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49.Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81%in shake flasks.Additionally,accumulation of 5-KGA was decreased by 63.52%with the resulting G.japonicas-Δga5dh-1-ga2dh-A strain.Using an intermittent fed-batch mode in a 3 L fermenter,2-KGA reached 235.3 g L^−1 with a 91.1%glucose conversion rate.Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L^−1 h^−1,11.99%higher than in the 3 L fermenter,and D-GA and 5-KGA by-products were completely converted to 2-KGA.

Current progress and prospects of enzyme technologies in future foods

Enzyme technologies are widely used in the food industry due to their advantages of high efficiency,specificity,and safety.Recently,“future foods”is emerging as a new research hotspot with healthier foods that are more nutritious,delicious,and sustainable;however,these foods still have problems with texture,nutrition,and flavor.Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects.In this review,we summarize enzyme technology applications in future food production,focusing on food texture,safety,and flavors.Furthermore,we discuss the prospects of enzyme-based technologies for future food production,including the modification of enzyme activities,the development of suitable food-grade hosts for enzyme production,and the optimiza-tion synergistic multi-enzyme systems.