Growth-coupled evolution of phosphoketolase to improve l-glutamate production by Corynebacterium glutamicum

Dele-Osibanjo, Taiwo; Li, Qinggang; Zhang, Xiaoli; Guo, Xuan; Feng, Jinhui; Liu, Jiao; Sun, Xue; Wang, Xiaowei; Zhou, Wenjuan; Zheng, Ping; Sun, Jibin; Ma, Yanhe

doi:10.1007/s00253-019-10043-6

Cited by 16 publications

(6 citation statements)

References 52 publications

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“…For further exploiting the potential of these strategies, it might be promising to increase the enzyme efficiencies of the PK pathway. For instance, Dele-Osibanjo et al applied directed evolution methods to screen for positive mutations of the PK enzyme, and successfully improved l -glutamate production in Corynebacterium glutamicum …”

Section: Discussionmentioning

confidence: 99%

“…For instance, Dele-Osibanjo et al applied directed evolution methods to screen for positive mutations of the PK enzyme, and successfully improved L-glutamate production in Corynebacterium glutamicum. 40 Besides the ability to produce acetyl-CoA with less carbon loss, since the PK pathway consumes xylulose-5-phosphate in the ox-PPP, it also has the potential to thermodynamically pull more carbon flux through the PP pathway for 3-HP production. To push the carbon flux through the ox-PPP, we first tried overexpression of ZWF1 and GND1 in the PP pathway, but this had minor effect on 3-HP production (Figure 4).…”

Section: ■ Discussionmentioning

confidence: 99%

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Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production

Qin

et al. 2020

ACS Synth. Biol.

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The central carbon metabolite acetyl-CoA and the cofactor NADPH are important for the synthesis of a wide array of biobased products. Here, we constructed a platform yeast strain for improved provision of acetyl-CoA and NADPH, and used the production of 3-hydroxypropionic acid (3-HP) as a case study. We first demonstrated that the integration of phosphoketolase and phosphotransacetylase improved 3-HP production by 41.9% and decreased glycerol production by 48.1% compared with that of the control strain. Then, to direct more carbon flux toward the pentose phosphate pathway, we reduced the expression of phosphoglucose isomerase by replacing its native promoter with a weaker promoter, and increased the expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by replacing their native promoters with stronger promoters. This further improved 3-HP production by 26.4%. Furthermore, to increase the NADPH supply we overexpressed cytosolic aldehyde dehydrogenase, and improved 3-HP production by another 10.5%. Together with optimizing enzyme expression of acetyl-CoA carboxylase and malonyl-CoA reductase, the final strain is able to produce 3-HP with a final titer of 864.5 mg/L, which is a more than 24-fold improvement compared with that of the starting strain. Our strategy combines the PK pathway with the oxidative pentose phosphate pathway for the efficient provision of acetyl-CoA and NADPH, which provides both a higher theoretical yield and overall yield than the reported yeast-based 3-HP production strategies via the malonyl-CoA reductase-dependent pathway and sheds light on the construction of efficient platform cell factories for other products.

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Section: Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production

Qin

et al. 2020

ACS Synth. Biol.

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“…4a, xfp weakens the Embden-Meyerhof-Parnas (EMP) pathway during expression, converting one molecule of glucose into three molecules of acetyl-CoA without CO 2 emissions. The theoretical carbon yield of acetyl-CoA increased from 0.66 mol mol mol − 1 to 1.00 mol mol mol − 1 [23], and carbon is totally preserved in glycolytic metabolism, similar to acetyl-CoA. Enhanced carbon ux in the TCA cycle.…”

Section: Introducing the Nog Pathway To Reduce Carbon Loss And Increa...mentioning

confidence: 97%

Optimization of trans-4-hydroxyproline synthesis pathway by rearrangement center carbon metabolism in Escherichia coli

Gong

Wang

et al. 2023

Preprint

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Background Trans-4-hydroxyproline (T-4-HYP) is a promising intermediate in the synthesis of antibiotic drugs. However, its industrial production remains challenging due to the low production efficiency of T-4-Hyp.This study focused on designing the key nodes of anabolic pathway to enhance carbon flux and minimize carbon loss, thereby maximizing the production potential of microbial cell factories. Results First, a basic strain, HYP-1, was developed by releasing feedback inhibitors and expressing heterologous genes for the production of trans-4-hydroxyproline. Subsequently, the biosynthetic pathway was strengthened while inhibitory pathways were disrupted, resulting in increased metabolic flow of α-ketoglutarate in the Tricarboxylic acid cycle. The introduction of the NOG pathway rearranged the central carbon metabolism, redirecting glucose towards acetyl-CoA. Furthermore, the supply of NADH-reducing power was enhanced to improve the acid production capacity of the strain. Finally, the fermentation process of T-4-HYP was optimized using a continuous feeding method. The rate of sugar supplementation controlled the dissolved oxygen concentrations during fermentation, and Fe2+ was continuously fed to supplement the reduced iron for hydroxylation. These modifications ensured an effective supply of proline hydroxylase cofactors (O2 and Fe2+), enabling efficient production of T-4-HYP in the microbial cell factory system. The strain HYP-10 produced 89.4 g/L of T-4-HYP e in a 5L fermenter, with a total yield of 0.34 g/g, which is the highest reported T-4-HYP production so far. Conclusion This study presents a strategy for establishing a microbial cell factory capable of producing T-4-HYP at high levels, making it suitable for large-scale industrial production. Additionally, this study provides valuable insights into regulating Synthesis of other compounds with α-ketoglutaric acid as precursor.

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“…Corynebacterium glutamicum , a high-GC content, Gram-positive soil bacterium, is an important industrial microorganism for food safety, which is used to produce a variety of amino acids (Dele-Osibanjo et al 2019 ; Ghiffary et al 2022 ; Liu et al 2022b ; Yu et al 2021 ). Although l -proline is one of nonessential amino acids for organisms, it has been widely used in agriculture, feed industry, food industry, and pharmaceutical industry (Jensen 2013 ; Ren et al 2020 ; Zhang et al 2020 , 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid screening of point mutations by mismatch amplification mutation assay PCR

Zhang,

Liu,

Liu

et al. 2024

Appl Microbiol Biotechnol

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Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the l-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. Key points • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.

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Growth-coupled evolution of phosphoketolase to improve l-glutamate production by Corynebacterium glutamicum

Cited by 16 publications

References 52 publications

Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production

Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production

Optimization of trans-4-hydroxyproline synthesis pathway by rearrangement center carbon metabolism in Escherichia coli

Rapid screening of point mutations by mismatch amplification mutation assay PCR

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