Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

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.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles.It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics.To meet its growing commercial demands,we engineered Escherichia coli strains for the high-yield production of ectoine.The ectABC gene cluster from the native ectoine producer Halomonas elongata was intro-duced into different Escherichia coli(E.Coil)strains via plasmids and 0.8 g L^(-1)of ectoine was produced in flask cultures by engineered E.coli BL21(DE3).Subsequently,we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA,ectB,and ectC,which increased the ectoine yield to 1.6 g L^(-1).After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant(G1A,C932T)and the H.elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor,the titer of ectoine reached to 5.5 g L^(-1)in flask cultures.Finally,the engineered strain produced 60.7 g L^(-1)ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Recent advances and challenges in microbial production of human milk oligosaccharides

Human milk oligosaccharides(HMOs)are one of the major differences between livestock milk and human milk,and the prebiotic functions of HMOs have been verified through in vitro and clinical trials.The most abundant HMOs include 2′-fucysollactose(2′-FL),3-fucosyllactose(3-FL),lacto-N-neotetraose(LNnT)and lacto-N-tetraose(LNT);their application and synthesis have attracted wide attentions.In recent years,the biotechnological production of 2′-FL,3-FL,LNnT and LNT have emerged based on techniques such as whole-cell catalysis and fermentation.In particular,the development of metabolic engineering and synthetic biology methods and strategies have facilitated efficient biosynthesis of these HMOs.However,these advantages have not been systematically reviewed yet.In this review,we first discuss the structures and applications of HMOs;secondly,strategies of microbial synthesis of the most abundant 2′-FL,3-FL,LNnT and LNT are summarized and compared.Finally,challenges and perspectives of efficient microbial production of HMOs as well as strategies for overcoming the challenges are discussed.This review reveals the whole picture of recent development in HMOs microbial synthesis and can further facilitate the understanding of limiting factors,and further propose a few directions to promote the development of efficient production hosts.

Current advances in design and engineering strategies of industrial enzymes

Enzymes are considered as functional proteins,also known as biocatalysts,which are required for normal cellular function.The commercial production of enzymes with distinct features always remains the top priority of researchers for industrial biocatalysis.However,the insufficient yield,low stability,low activity,formation of by-products,complex purification pro-cess,and many other factors are challenging for the enzyme production industry.Novel advancements in protein engineering,especially the combination of multidisciplinary techniques including post-translational enzyme modification,structured assisted protein tailoring and computational modeling approaches,open a new horizon to more efficient production of bio-catalysts.In this review,we focus on the development process of enzyme modification design strategies,such as rational design,semi-rational design and de novo design.Selected examples for each strategy,which are particularly useful for novel enzyme design,are given.We hope this review is helpful for the development of biocatalysts for industrial application.

Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose(LNnT)

Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.

Statistical modeling and optimization for enhanced hyaluronic acid production by batch culture of Sreptococcus zooepidemicus via the supplement of uracil

This work is aimed to achieve the optimal hyaluronic acid(HA)production by batch culture of Streptococcus zooepidemicus via the supplement of nucleotide bases using response surface methodology(RSM).First,the influence of nucleotide bases(adenine,guanine,cytosine,thymine,and uracil)on microbial HA production was investigated using fractional factorial design(FFD).By a 2^(5-2)FFD,uracil was found to be the most significant factor for cell growth and HA production,while the other nucleotide bases were shown to have no significant effects on cell growth and HA production.Also,the impact of uracil on cell growth and HA production was further investigated by RSM,where two variables were considered:uracil concentration and supplement time.The optimal uracil concentration and supplement time were found to be 0.051 g/L and 7 h,respectively,and the predicted maximal HA production reached 6.42 g/L.The maximal HA production increased from 5.0 g/L of the control without uracil supplement to 6.31 g/L at the optimal conditions in validation experiments.

Enzymatic production of N‑acetylneuraminic acid:advances and perspectives

N-Acetyl-d-neuraminic acid(NeuAc),a well-known and well-studied sialic acid,is found in cell surface glycolipids and glycoproteins,where it performs a variety of biological functions.The use of NeuAc as a nutraceutical for infant brain development and as an intermediate for pharmaceutical production demands its production on an industrial scale.Natural extraction,chemical synthesis,enzymatic synthesis,and biosynthesis are the methods used for NeuAc production.Among these methods,enzymatic synthesis using N-acetyl-glucosamine(GlcNAc)2-epimerase(AGE)for epimerization and N-acetyld-neuraminic acid lyase(NAL)for aldol condensation,has been reported to produce NeuAc with high production efciency.In this review,we discuss advances in the two-step enzymatic synthesis of NeuAc using pyruvate and GlcNAc as substrates.The major challenges in producing NeuAc with high yield are highlighted,including multiple parameter-dependent processes,undesirable reversibility,and diminished solubility of AGEs and NALs.Further,diferent strategies applied to overcome the limitations of the two-step enzymatic production are discussed,such as pyruvate concentration and temperature shift during the process to increase conversion yield,use of mathematical and computational simulations for process optimization,enzyme engineering to make enzymes highly efcient,and the use of tags and chaperones to increase enzyme solubility.We suggest future directions and the strategies that can be followed to improve enzymatic synthesis of NeuAc.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Gene regulation by trans-acting small RNAs(sRNAs)has considerable advantages over other gene regulation strategies.However,synthetic sRNAs mainly take natural sRNAs(MicC or SgrS)as backbones and comprise three functional elements folding into two or more stem-loop structures:an mRNA base pairing region,an Hfq-binding structure,and a rho-independent terminator.Due to limited numbers of natural sRNAs and complicated backbone structures,synthetic sRNAs suffer from low activity programmability and poor structural modularity.Moreover,natural sRNA backbone sequences may increase the possibility of unwanted recombination.Here,we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs(ssl-sRNAs),which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons.Particularly,ssl-sRNA requires no independent Hfq-binding structure,as the polyU tail fulfills the roles of binding Hfq.A thermodynamic-based scoring model and a web server sslRNAD(http://www.kangzlab.cn/)were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities.ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs.The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis.A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries.As examples,the E.coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs.ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

In silico cell factory design driven by comprehensive genome‑scale metabolic models:development and challenges

Genome-scale metabolic models(GEMs)have been widely used to design cell factories in silico.However,initial flux balance analysis only considers stoichiometry and reaction direction constraints,so it cannot accurately describe the distribution of metabolic flux under the control of various regulatory mechanisms.In the recent years,by introducing enzymology,thermodynamics,and other multiomics-based constraints into GEMs,the metabolic state of cells under different conditions was more accurately simulated and a series of algorithms have been presented for microbial phenotypic analysis.Herein,the development of multiconstrained GEMs was reviewed by taking the constraints of enzyme kinetics,thermodynamics,and transcriptional regulatory mechanisms as examples.This review focused on introducing and summarizing GEMs application tools and cases in cell factory design.The challenges and prospects of GEMs development were also discussed.

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions.However,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue.In this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask cultures.Afterwards,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic efficiencies.On this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch cultures.Moreover,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.

多学科交叉发酵工程复合型研究生培养

发酵工程是利用对微生物或其他生物细胞进行改造,在特定的生物反应器内,培养生产某种特定产品的工业化生产过程和技术体系。发酵工程从纯粹依赖经验积累的古老的食品发酵,发展成为食品、农业、医药、化工等生产生活资料的重要生产方式,成为支撑人类可持续发展的关键技术,这离不开交叉学科技术的持续进步。多学科融合交叉和我国在全球产业链的不断上移,必然对新形势下发酵工程复合型人才培养提出更高要求。为不断完善多学科交叉的发酵工程复合型人才培养体系,近年来,研究室不断凝练与提升人才培养理念,积极深化人才培养体系改革。围绕培养方案、招生体系、师资背景、课题设置、科研实践、评价体系等方面展开了系统的研究和实践,推动了发酵工程和相关支撑行业的技术进步,为培养具有学科交叉知识的复合型人才,进而为我国从发酵大国向发酵强国的转变贡献了重要力量。

洋河浓香型白酒发酵过程酒醅微生物群落结构解析及其与有机酸合成的相关性

【目的】解析江苏洋河酒厂浓香型白酒窖内发酵过程酒醅微生物群落结构,建立酒醅微生物与主要有机酸合成的关联性。【方法】通过宏基因组测序获得白酒发酵过程中微生物群落结构变化规律,利用主成分分析和偏最小二乘回归分析寻找酒醅中影响主要有机酸合成的关键微生物。【结果】根据微生物组成结构变化和有机酸合成变化规律,可将白酒窖内发酵分为两个时期(0–14 d和15–60 d)。其中窖内发酵0–15 d与主要有机酸合成相关的微生物数量显著高于15–60 d的。窖内发酵过程与主要有机酸合成相关的微生物包括7个菌属,分别为乳杆菌属(Lactobacillus)、葡萄球菌属(Staphylococcus)、酵母属(Saccharomyces)、Naumovozyma、伊萨酵母属(Issatchenkia)、嗜冷芽孢杆菌属(Psychrobacillus)和根霉属(Rhizopus)。【结论】本研究识别了白酒窖内发酵过程中与主要有机酸合成相关的核心和关键微生物,可为阐明白酒窖内发酵产酸机理和保障白酒品质的稳定性奠定研究基础和理论依据。

动物细胞培养技术在人造肉研究中的应用

人造肉作为2018年全球十大突破和新兴科技之一,因其来源可追溯、食品安全性和绿色可持续等优势得到广泛的关注。欧美等国家已经投入大量资源开展细胞培养人造肉研究,未来将对我国的肉制品及食品市场造成一定的冲击。现阶段,细胞培养人造肉生产的挑战在于如何高效模拟动物肌肉组织生长环境,并在生物反应器中实现大规模的生产。尽管动物细胞组织培养技术已经得到深入的研究,并取得了不同程度的成功应用,但由于现有动物细胞组织培养成本与技术要求较高,仍不能实现大规模的产业化培养。因此,对于人造肉的生产来说,开发高效、安全的大规模细胞培养技术是亟需解决的问题,可以有效降低生产成本,实现产业化应用。文中通过介绍基于人造肉生物制造的动物细胞组织培养技术研究现状,具体阐述了目前的挑战和关键技术问题,并初步探讨了其可能的解决策略和应用前景。

细胞培养肉规模化生产工艺及反应器展望

体外培养动物肌肉组织作为食用材料的构想在20世纪30年代就有了,90年代末还出现了相关的专利,但该技术到目前为止还没有大规模生产的例子。过去五、六年来,尤其是2013年世界首个细胞培养牛肉汉堡公开试吃活动以后,培养肉(Cultured meat)被大量报道。截至2019年3月,全世界已有超过25家公司宣布正在研究将培养肉推向市场,但仍没有一家企业能把产品放到普通消费者面前。这其中一部分限制因素是监管和社会伦理方面的争议,但更大的问题是生产成本居高不下。规模化生产是降低单位成本的有效手段,但目前业界对动物细胞大规模培养的传质、传热、混合、剪切应力等问题还缺乏足够关注。动物细胞培养传统上多用于量低价高的医疗、医药领域,提高产量的途径往往是多组体积不超过2?20m^3的设备并列运行,保持培养环境均一性问题不大。但人工培养动物肌肉细胞若作为大宗食品进入市场与传统养殖业进行有效竞争,所需的生物反应器规模和细胞密度还需在目前常用的动物细胞培养技术基础上提高至少一个数量级。因此,培养肉大规模培养工艺与设备的进步是使其成为替代性动物蛋白来源的前提条件。本工作对培养肉规模化生产能采用的反应器类型、操作模式、存在的主要问题及与反应器相关的研究方向进行探讨,为国内相关企业和科研机构提供参考。