Dataset Information

Engineering Corynebacterium glutamicum triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate.

ABSTRACT:

Background

Lignocellulose biomass contains high amount of biotin and resulted in an excessive biotin condition for cellulosic glutamic acid accumulation by Corynebacterium glutamicum. Penicillin or ethambutol triggers cellulosic glutamic acid accumulation, but they are not suitable for practical use due to the fermentation instability and environmental concerns. Efficient glutamic acid production from lignocellulose feedstocks should be achieved without any chemical inductions.

Results

An industrial strain C. glutamicum S9114 was metabolically engineered to achieve efficient glutamic acid accumulation in biotin-excessive corn stover hydrolysate. Among the multiple metabolic engineering efforts, two pathway regulations effectively triggered the glutamic acid accumulation in lignocellulose hydrolysate. The C-terminal truncation of glutamate secretion channel MscCG (?C110) led to the successful glutamic acid secretion in corn stover hydrolysate without inductions. Then the ?-oxoglutarate dehydrogenase complex (ODHC) activity was attenuated by regulating odhA RBS sequence, and glutamic acid accumulation was further elevated for more than fivefolds. The obtained C. glutamicum XW6 strain reached a record-high titer of 65.2 g/L with the overall yield of 0.63 g/g glucose using corn stover as the starting feedstock without any chemical induction.

Conclusions

Metabolic engineering method was successfully applied to achieve efficient glutamic acid in biotin-rich lignocellulose hydrolysate for the first time. This study demonstrated the high potential of glutamic acid production from lignocellulose feedstock.

SUBMITTER: Wen J

PROVIDER: S-EPMC6463653 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Engineering <i>Corynebacterium glutamicum</i> triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate.

Wen Jingbai J Bao Jie J

Biotechnology for biofuels 20190415

<h4>Background</h4>Lignocellulose biomass contains high amount of biotin and resulted in an excessive biotin condition for cellulosic glutamic acid accumulation by <i>Corynebacterium glutamicum</i>. Penicillin or ethambutol triggers cellulosic glutamic acid accumulation, but they are not suitable for practical use due to the fermentation instability and environmental concerns. Efficient glutamic acid production from lignocellulose feedstocks should be achieved without any chemical inductions.<h4 ...[more]

PMID: 31011369

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Project description:BackgroundLignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock.ResultsWe identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum. Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 μg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment.ConclusionsThe high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients for cellulosic fermentations.

Project description:The production of value-added chemicals alongside biofuels from lignocellulosic hydrolysates is critical for developing economically viable biorefineries. Here, the production of propionic acid (PA), a potential building block for C3-based chemicals, from corn stover hydrolysate is investigated using the native PA-producing bacterium Propionibacterium acidipropionici.A wide range of culture conditions and process parameters were examined and experimentally optimized to maximize titer, rate, and yield of PA. The effect of gas sparging during fermentation was first examined, and N2 was found to exhibit improved performance over CO2. Subsequently, the effects of different hydrolysate concentrations, nitrogen sources, and neutralization agents were investigated. One of the best combinations found during batch experiments used yeast extract (YE) as the primary nitrogen source and NH4OH for pH control. This combination enabled PA titers of 30.8 g/L with a productivity of 0.40 g/L h from 76.8 g/L biomass sugars, while successfully minimizing lactic acid production. Due to the economic significance of downstream separations, increasing titers using fed-batch fermentation was examined by changing both feeding media and strategy. Continuous feeding of hydrolysate was found to be superior to pulsed feeding and combined with high YE concentrations increased PA titers to 62.7 g/L and improved the simultaneous utilization of different biomass sugars. Additionally, applying high YE supplementation maintains the lactic acid concentration below 4 g/L for the duration of the fermentation. Finally, with the aim of increasing productivity, high cell density fed-batch fermentations were conducted. PA titers increased to 64.7 g/L with a productivity of 2.35 g/L h for the batch stage and 0.77 g/L h for the overall process.These results highlight the importance of media and fermentation strategy to improve PA production. Overall, this work demonstrates the feasibility of producing PA from corn stover hydrolysate.

Project description:To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 ?g of biotin per liter for normal growth, the bioY disruptant (?bioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ?bioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ?bioY strain. By selectively using the resulting two strains (?bioB and ?bioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 ?g to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 ?g per liter).

Project description:BackgroundEfficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources.ResultsWe monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate.ConclusionsGrowth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.

Project description:Background:Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results:In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (?ldhA?pta?pqo?catPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L-1 h-1 during the anaerobic stage. Conclusion:This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.