谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

Microbial production of 4-amino-1-butanol,a four-carbon amino alcohol

4-Amino-1-butanol (4AB) serves as an important intermediate compound for drugs and a precursor of biodegradable polymers used for gene delivery. Here, we report for the first time the fermentative production of 4AB from glucose by metabolically engineered Corynebacterium glutamicum harboring a newly designed pathway comprising a putrescine (PUT) aminotransferase (encoded by ygjG) and an aldehyde dehydrogenase (encoded by yqhD) from Escherichia coli, which convert PUT to 4AB. Application of several metabolic engineering strategies such as fine-tuning the expression levels of ygjG and yqhD, eliminating competing pathways, and optimizing culture condition further improved 4AB production. Fed-batch culture of the final metabolically engineered C. glutamicum strain produced 24.7 g/L of 4AB. The strategies reported here should be useful for the microbial production of primary amino alcohols from renewable resources.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

High‐titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum

Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied M_w. The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA‐hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal M_w was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

D-lactic acid production by a genetically engineered strain <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Based on its ability to produce lactic acid from glucose in mineral salt medium under anaerobic conditions, genetic modifications on Corynebacterium glutamicum Res 167 were carried out with the aim of producing optical pure D-lactic acid, involving the knockout of L-lactate dehydrogenase gene from C. glutamicum and the heterologous expression of D-lactate dehydrogenase gene from Lactobacillus bulgaricus into C. glutamicum. D-lactic acid production of the genetically engineered strain C. glutamicum Res 167Δldh/ldhA was 17.92 g/l (optical purity higher than 99.9%) after 16 h fermentation, which was 32.25% higher than the lactic acid production of the parental strain.     

Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition

Capture and conversion of CO₂ to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry‐relevant chemicals from CO₂. However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO₂ to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP‐driven malonyl‐CoA synthesis pathway and heterologous phosphoketolase (PHK)‐phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl‐CoA directly from CO₂ in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl‐CoA‐derived biochemicals.     

Metabolic engineering of Pichia pastoris for malic acid production from methanol

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value‐added chemicals from methanol.     

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production

The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l⁻¹). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC_Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN_Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l⁻¹ after 12-h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high-temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.     

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.     

A “plug‐n‐play” modular metabolic system for the production of apocarotenoids

Apocarotenoids, such as α‐, β‐ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β‐ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli‐based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N‐terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α‐ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α‐ionone in fed‐batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re‐engineered to produce 32 mg/L and 500 mg/L of β‐ionone in flask and bioreactor, respectively (>80‐fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the “plug‐n‐play” modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes.     

Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

Microbial production of 1,5-diaminopentane (DAP) from renewable feedstock is a promising and sustainable approach for the production of polyamides. In this study, we constructed a β-glucosidase (BGL)-secreting Corynebacterium glutamicum and successfully used this strain to produce DAP from cellobiose and glucose. First, C. glutamicum was metabolically engineered to produce l -lysine (a direct precursor of DAP), followed by the coexpression of l -lysine decarboxylase and BGL derived from Escherichia coli and Thermobifida fusca YX (Tfu0937), respectively. This new engineered C. glutamicum strain produced 27 g/L of DAP from cellobiose in CGXII minimal medium using fed-batch cultivation. The yield of DAP was 0.43 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose), which is the highest yield reported to date. These results demonstrate the feasibility of DAP production from cellobiose or cellooligosaccharides using an engineered C. glutamicum strain.     

Metabolic engineering of 1,2-propanediol pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We analyzed 1,2-propanediol (1,2-PD) production in metabolically engineered Corynebacterium glutamicum. Wild-type C. glutamicum produced 93 μM 1,2-PD after 132 h incubation under aerobic conditions. No gene encoding the methylglyoxal synthase (MGS) which catalyzes the first step of 1,2-PD synthesis from the glycolytic pathway was detected on the C. glutamicum genome, but several genes annotated as encoding putative aldo-keto reductases (AKRs) were present. AKR functions as a methylglyoxal reductase in the 1,2-PD synthesis pathway. Expressing Escherichia coli mgs gene in C. glutamicum increased 1,2-PD yield 100-fold, suggesting that wild-type C. glutamicum carries the genes downstream of MGS in the 1,2-PD synthesis pathway. Furthermore, simultaneous overexpression of mgs and cgR_2242, one of the genes annotated as AKRs, enhanced 1,2-PD production to 24 mM. This work establishes that 1,2-PD synthesis by C. glutamicum, previously unknown, is possible.     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.