使用动态分子开关调控大肠杆菌生产莽草酸

莽草酸是大肠杆菌合成芳香族氨基酸的中间代谢物,也是抗流感药物"达菲"的重要合成前体。合成莽草酸需要截断莽草酸途径,导致芳香族氨基酸无法合成,因此面临细胞生长受到抑制的问题。使用动态调控策略通过将细胞生长和莽草酸的合成相互分离,可以提高菌株的生产性能。通过使用生长偶联型启动子和降解决定子(Degrons),组建动态分子开关。利用该动态分子开关实现细胞生长与莽草酸合成分离,在5L发酵罐中经过72h发酵得到了14.33g/L的莽草酸。结果表明,这种动态分子开关可以通过调控靶蛋白丰度来改变碳流量平衡,使菌株获得更优秀的生产性能。     

Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control

Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.     

Enhanced protein and biochemical production using CRISPRi-based growth switches

Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45 h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals.     

Metabolic modeling and response surface analysis of an Escherichia coli strain engineered for shikimic acid production

Background

Classic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media.

Results

The obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process.

Conclusions

The combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.

     

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82 g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53 g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.     

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.     

Production of microbial lipid by Cryptococcus curvatus on rice straw hydrolysates

Microbial biolipids/biodiesels derived from volatile fatty acids (VFAs) can be a valuable alternative to plant oils if optimum fermentation conditions are determined. VFAs were used for cell mass and microbial lipid production by Cryptococcus curvatus. The lipid content in the cells increased up to 48% and 28% in batch cultures with the use of 20 g/L glucose and 6 g/L of VFAs as the carbon source, respectively. In this study, C. curvatus used VFAs as a carbon source via anaerobic digestion of rice straw hydrolysates. VFAs produced from rice straw resulted in yield of 0.43 g VFAs/g substrate and 40% higher specific growth rate(0.305 h⁻¹) than synthetic VFAs. The highest fatty acid composition observed was C18:1, was obtained using glucose and VFAs as the carbon source to yield a cetane number of 56–59, which is suitable for biodiesel production. The cost of microbial lipids was estimated to be 0.30–1.15 USD/L given 0–150 USD/ton of VFAs cost for a yield of 0.17 g/g of lipids. Thus, VFAs can be a suitable carbon source for economical biodiesel production.     

Development of a growth coupled and multi-layered dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin biosynthesis in Escherichia coli

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.     

Studies on the production of shikimic acid using the <Emphasis Type="Italic">aroK</Emphasis> knockout strain of <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu^®) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.     

Establishing an Autonomous Cascaded Artificial Dynamic (AutoCAD) regulation system for improved pathway performance

Endogenous metabolic pathways in microbial cells are usually precisely controlled by sophisticated regulation networks. However, the lack of such regulations when introducing heterologous pathways in microbial hosts often causes unbalanced enzyme expression and carbon flux distribution, hindering the construction of highly efficient microbial biosynthesis systems. Here, using naringenin as the target compound, we developed an Autonomous Cascaded Artificial Dynamic (AutoCAD) regulation system to automatically coordinate the pathway expression and redirect carbon fluxes for enhanced naringenin production. The AutoCAD regulation system, consisting of both intermediate-based feedforward and product-based feedback control genetic circuits, resulted in a 16.5-fold increase in naringenin titer compared with the static control. Fed-batch fermentation using the strain with AutoCAD regulation further enhanced the naringenin titer to 277.2 mg/L. The AutoCAD regulation system, with intermediate-based feedforward control and product-triggered feedback control, provides a new paradigm of developing complicated cascade dynamic control to engineer heterologous pathways.     

Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynthesis pathway

Hyaluronic acid (HA) production in Streptococcus zooepidemicus competes for the carbon source along with biomass formation, lactate formation (via glycolysis) and pentose phosphate pathway (PPP). In our studies, increase in HA molecular weight was observed by redirecting the carbon flux towards HA biosynthesis pathway by partially inhibiting the glycolytic pathway. Batch bioreactor (1.2 L) studies showed that with the addition of 25 μM sodium iodoacetate, 5 g/L tryptophan and 10 g/L pyruvate, which are glycolytic inhibitors, HA molecular weight increased to 3.2, 3.2 and 3.1 MDa respectively compared to control run (2.4 MDa). Yield coefficients Y_HA/S and Y_LA/S showed inverse relationship, indicating competition for glucose between HA and lactic acid formation. Addition of 5 g/L glutamine along with 25 μM sodium iodoacetate also increased the HA concentration to 5.0 g/L from 2.0 g/L in control run. Metabolic flux analysis studies show that concentration and molecular weight of HA is increased by decreasing carbon flux towards glycolysis and PPP and increasing carbon flux towards HA precursor formation. It was observed that specific growth rate of the cells correlated positively to the specific HA production rate and negatively to the molecular weight of HA produced. Addition of antioxidant tannic acid also increased molecular weight to 3.0 MDa.     

Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone

(+)-Nootkatone is a valuable, functional sesquiterpene that is widely used in food, cosmetics, pharmaceutical, agriculture, and other fields. However, only traces of it accumulate in plants, which is insufficient to meet the market demand. Therefore, commercial (+)-nootkatone is currently synthesized from (+)-valencene. Here, we engineered Saccharomyces cerevisiae to achieve high production of (+)-valencene. Employing gene screening, protein engineering and biosynthetic pathway optimization, we achieved 12.4 g/L (+)-valencene production with the mutant strain. This titer was further increased to 16.6 g/L, the highest titer reported to date, by coupling critical factors for cell growth and biochemical pathway induction. Subsequently, (+)-nootkatone was chemically synthesized from bio-fermented (+)-valencene with a yield of 80%. This study achieved efficient microbial synthesis of (+)-valencene, which may be utilized in industrial production and stabilize the supply of (+)-nootkatone.     

The mixed culture of microalgae Chlorella pyrenoidosa and yeast Yarrowia lipolytica for microbial biomass production

Microbial biomass which mostly generated from the microbial processes of bacteria, yeasts, and microalgae is an important resource. Recent concerns in microbial biomass production field, especially microbial lipid production for biofuel, have been focused towards the mixed culture of microalgae and yeast. To more comprehensive understanding of the mixed culture for microbial biomass, mono Chlorella pyrenoidosa, mono Yarrowia lipolytica and the mixed culture were investigated in the present work. Results showed that the mixed culture achieved significantly faster cell propagation of microalga and yeast, smaller individual cell size of yeast and higher relative chlorophyll content of microalga. The mixed culture facilitated the assimilation of carbon and nitrogen and drove the carbon flow to carbohydrate. Besides higher lipid yield (0.77 g/L), higher yields of carbohydrates (1.82 g/L), protein (1.99 g/L) and heating value (114.64 kJ/L) indicated the microbial biomass harvested from the mixed culture have more potential utilization in renewable energy, feedstuff, and chemical industry.

     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content.Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.     

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.