Plug-in repressor library for precise regulation of metabolic flux in Escherichia coli

In metabolic engineering, enhanced production of value-added chemicals requires precise flux control between growth-essential competing and production pathways. Although advances in synthetic biology have facilitated the exploitation of a number of genetic elements for precise flux control, their use requires expensive inducers, or more importantly, needs complex and time-consuming processes to design and optimize appropriate regulator components, case-by-case. To overcome this issue, we devised the plug-in repressor libraries for target-specific flux control, in which expression levels of the repressors were diversified using degenerate 5′ untranslated region (5’ UTR) sequences employing the UTR Library Designer. After we validated a wide expression range of the repressor libraries, they were applied to improve the production of lycopene from glucose and 3-hydroxypropionic acid (3-HP) from acetate in Escherichia coli via precise flux rebalancing to enlarge precursor pools. Consequently, we successfully achieved optimal carbon fluxes around the precursor nodes for efficient production. The most optimized strains were observed to produce 2.59 g/L of 3-HP and 11.66 mg/L of lycopene, which were improved 16.5-fold and 2.82-fold, respectively, compared to those produced by the parental strains. These results indicate that carbon flux rebalancing using the plug-in library is a powerful strategy for efficient production of value-added chemicals in E. coli.     

Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via β-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO₂ as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source

Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H₂ + CO₂. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H₂ + CO₂ using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.     

Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli

The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25 g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460 ± 10 mM (41.5 ± 1.1 g/L) in 48 h with a yield of 31 % and a productivity of 0.86 ± 0.05 g/L h. In contrast, the recombinant E. coli W3110 produced only 180 ± 8.5 mM 3-HP (15.3 ± 0.8 g/L) in 48 h with a yield and productivity of 26 % and 0.36 ± 0.02 g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Metagenomic analysis for the microbial consortium of anaerobic CO oxidizers

Metagenomics analysis has been applied to identify the dominant anaerobic microbial consortium of the carbon monoxide (CO) oxidizers in anaerobic sludge. Reads from the hypervariable V6 region in the bacterial 16s rDNA were aligned and finally clustered into operational taxonomic units (OTUs). The OTUs from different stages in anaerobic CO condition were classified. Alphaproteobacteria, clostridia, betaproteobacteria and actinobacteria were the most abundant groups, while alphaproteobacteria, betaproteobacteria and actinobacteria were variable groups. CO consumption and production efficiency of the microbial consortium were studied. Semi-continuous trials showed that these anaerobic CO oxidizers formed a stable microbial community, and the CO conversion rate was at over 84%, with the highest CO consumption activity of 28.9 mmol CO/g VSS●day and methane production activity at 7.6 mmol CH₄/g VSS●day during six cycles.     

Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid

Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B₁₂ under aerobic conditions. Recently, recombinant strains of P. denitrificans overexpressing a vitamin B₁₂-dependent glycerol dehydratase (DhaB) were developed to produce 3-hydroxypropionic acid (3-HP) from glycerol. The recombinant P. denitrificans could produce 3-HP successfully under aerobic conditions without an exogenous supply of vitamin B₁₂, but the 3-HP produced disappeared during extended cultivation due to the 3-HP degradation activity in this strain. This study developed mutant strains of P. denitrificans that do not degrade 3-HP. The following eight candidate enzymes, which might be responsible for 3-HP degradation, were selected, cloned, and studied for their activity in Escherichia coli: four (putative) 3-hydroxyisobutyrate dehydrogenases (3HIBDH), a putative 3-HP dehydrogenase (3HPDH), an alcohol dehydrogenase (ADH), and two choline dehydrogenases (CHDH). Among them, 3HIBDHI, 3HIBDHIV, and 3HPDH exhibited 3-HP degrading activity when expressed heterologously in E. coli. When 3hpdh alone or along with 3hibdhIV were disrupted from P. denitrificans, the mutant P. denitrificans exhibited greatly reduced 3-HP degradation activity that could not grow on 3-HP as the sole carbon and energy source. When the double mutant P. denitrificans Δ3hpdhΔ3hibdhIV was transformed with DhaB, an improved 3-HP yield (0.78 mol/mol) compared to that of the wild-type counterpart (0.45 mol/mol) was obtained from a 24-h flask culture. This study indicates that 3hpdh and 3hibdhIV (to a lesser extent) are mainly responsible for 3-HP degradation in P. denitrificans and their deletion can prevent 3-HP degradation during its production by recombinant P. denitrificans.     

Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO₂ directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L⁻¹ (348.8 mg/g dry cell weight) 3-HP directly from CO₂ in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO₂ in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO₂ fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.     

Elongation Factor P is Dispensable in Escherichia coli and Pseudomonas aeruginosa

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical from which a panel of chemicals can be generated. Klebsiella pneumoniae has been regarded as a promising host strain in glycerol-based 3-HP production for its exceptional ability to metabolize glycerol. Since the glycerol dissimilation mechanism governs the carbon flux distribution from glycerol, inducible strong promoters were usually employed to enhance the glycerol consumption and 3-HP production. Here, we report an alternative strategy that the native promoter of dhaB gene was applied to enhance 3-HP production in K. pneumoniae. The key enzyme genes (ald4 and dhaB) for 3-HP biosynthesis were co-expressed under this promoter. Metabolic analysis revealed that the 3-HP formation was partially coupled with cell metabolism. To optimize the production of 3-HP, the effects of glucose as energy source assistant were investigated based on the analysis of fermentation process kinetics. The highest 3-HP yield (3.77 g/L in flask) was observed upon optimized conditions. Since there were no additional inducers needed, the strategy of employing native promoter seems more feasible to industrial application. More importantly, the employment of constitutive promoter demonstrated an effective approach for decoupling the natural correlation between respiratory metabolism and glycerol dissimilation in K. pneumoniae.     

Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source

Klebsiella pneumoniae is a Gram-negative facultative anaerobe that metabolizes glycerol efficiently under both aerobic and anaerobic conditions. This microbe is considered an outstanding biocatalyst for transforming glycerol into a variety of value-added products. Crude glycerol is a cheap carbon source and can be converted by K. pneumoniae into useful compounds such as lactic acid, 3-hydroxypropionic acid, ethanol, 1,3-propanediol, 2,3-butanediol, and succinic acid. This review summarizes glycerol metabolism in K. pneumoniae and its potential as a microbial cell factory for the production of commercially important acids and alcohols. Although many challenges remain, K. pneumoniae is a promising workhorse when glycerol is used as the carbon source.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Metabolic engineering of Escherichia coli for the production of succinate from glycerol

Glycerol has become an ideal feedstock for the microbial production of bio-based chemicals due to its abundance, low cost, and high degree of reduction. We have previously reported the pathways and mechanisms for the utilization of glycerol by Escherichia coli in minimal salts medium under microaerobic conditions. Here we capitalize on such results to engineer E. coli for the production of value-added succinate from glycerol. Through metabolic engineering of E. coli metabolism, succinate production was greatly elevated by (1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate and (2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). As such, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). Our biocatalysts demonstrated a maximum specific productivity of ～400 mg succinate/gcell/h and a yield of 0.69 g succinate/g glycerol, on par with the use of glucose as a feedstock.     

Growth of Eubacterium limosum with Carbon Monoxide as the Energy Source

Eubacterium limosum grew with CO as the sole source of energy and formed acetate and CO₂ as the major products. The generation time on CO was 7 h. Uninhibited growth occurred in cultures containing 50% CO or less, but growth occurred at all concentrations tested (i.e., up to 75% CO). The pH optimum for growth was 7.0 to 7.2, whereas growth was poor at a pH below 6.7. CO₂ stimulated growth on CO. CO was preferentially utilized when both CO and H₂ were present.     

3个植物源苯甲醇酰基转移酶合成乙酸肉桂酯的研究

[背景]乙酸肉桂酯是一种重要的香料化合物,在化妆品和食品工业上具有广泛的应用,传统的生产方法主要依靠植物提取和化学合成。[目的]通过筛选不同植物源的酰基转移酶,利用大肠杆菌从头合成乙酸肉桂酯。[方法]首先,通过在苯丙氨酸高产菌BPHE中表达异源基因苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase from Arabidopsis thaliana,AtPAL)、对羟基肉桂酰辅酶A连接酶(Hydroxycinnamate:CoA Ligase from Petroselinum crispum,Pc4CL)和肉桂酰辅酶 A 还原酶(Cinnamyl-CoA Reductase from Arabidopsis thaliana,AtCCR),并结合大肠杆菌自身的内源性醇脱氢酶(Alcohol Dehydrogenases,ADHs)或醛酮还原酶(Aldo-Keto Reductases,AKRs)的催化作用构建了从苯丙氨酸到肉桂醇的生物合成途径。然后,苯甲醇苯甲酰转移酶(Benzyl Alcohol O-Benzoyltransferase from Nicotiana tabacum,ANN09798;Benzyl Alcohol O-Benzoyltransferase from Clarkia breweri,ANN09796)或苯甲醇乙酰转移酶(Benzyl Alcohol Acetyltransferase from Clarkia breweri,BEAT)被引入到上述重组大肠杆菌中发酵培养生产乙酸肉桂酯。最后,在大肠杆菌中过表达乙酰辅酶A合成酶(Acetyl Coenzyme A Synthetase,ACS)来提高底物乙酰辅酶A的量。[结果]探讨了 3个植物源苯甲醇酰基转移酶生物合成乙酸肉桂酯的能力,并应用于合成乙酸肉桂酯的细胞工厂,最终使乙酸肉桂酯最高产量达到166.9±6.6mg/L。[结论]植物源苯甲醇酰基转移酶具有一定的底物宽泛性,能以肉桂醇为底物催化合成乙酸肉桂酯。首次利用植物源的苯甲醇酰基转移酶合成乙酸肉桂酯,为微生物细胞工厂以葡萄糖作为碳源生产乙酸肉桂酯提供参考。     

Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering

The overexpression of acetyl-CoA (CoA) synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation. It also greatly enhanced acetate assimilation when acetate was used as a carbon source. These features are ideal for applications in metabolic engineering. ACS overexpression can be strategically applied to reduce acetate byproduct, recover wasted carbon, and redirect carbon flux toward more favorable pathways. The native acs gene was cloned and overexpressed in E. coli. Studies showed significant effects on acetate production and assimilation in cultures grown in minimal and complex media with glucose or acetate as the carbon source.