Effect of NADPH availability on free fatty acid production in <i>Escherichia coli</i>

Li, Wei; Wu, Hui; Li, Mai; San, Ka‐Yiu

doi:10.1002/bit.26464

Cited by 38 publications

(29 citation statements)

References 32 publications

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“…Since the lignocellulosic biomass can be hydrolyzed into two major sugars of glucose and xylose (Gawand et al, 2013), the strategy for the efficient consumption of both sugars is highly desirable for the production of useful chemicals with low cost. In the production of industrially important valueadded products such as 3-hydroxybutyrate (3HB), methyl 3hydoxybutyrate (MHB), amino acids, fatty acids, and isoprenoids (Siedler et al, 2011;Perez-Zabaleta et al, 2016;Yanase et al, 2016;Niu et al, 2017;Li et al, 2018), NADPH is the essential molecule, and thus its availability remains a major hurdle for the efficient production of useful chemicals and fuels (Spaans et al, 2015). Several metabolic engineering strategies have, therefore, been considered, and tested in practice.…”

Section: Introductionmentioning

confidence: 99%

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

Matsuoka

Kurata

2020

Front. Bioeng. Biotechnol.

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Lignocellulosic biomass can be hydrolyzed into two major sugars of glucose and xylose, and thus the strategy for the efficient consumption of both sugars is highly desirable. NADPH is the essential molecule for the production of industrially important valueadded chemicals, and thus its availability is quite important. Escherichia coli mutant lacking the pgi gene encoding phosphoglucose isomerase (Pgi) has been preferentially used to overproduce the NADPH. However, there exists a disadvantage that the cell growth rate becomes low for the mutant grown on glucose. This limits the efficient NADPH production, and therefore, it is quite important to investigate how addition of different carbon source such as xylose (other than glucose) effectively improves the NADPH production. In this study, we have developed a kinetic model to propose an efficient NADPH production system using E. coli pgi-knockout mutant with a mixture of glucose and xylose. The proposed system adds xylose to glucose medium to recover the suppressed growth of the pgi mutant, and determines the xylose content to maximize the NADPH productivity. Finally, we have designed a mevalonate (MVA) production system by implementing ArcA overexpression into the pgi-knockout mutant using a mixture of glucose and xylose. In addition to NADPH overproduction, the accumulation of acetyl-CoA (AcCoA) is necessary for the efficient MVA production. In the present study, therefore, we considered to overexpress ArcA, where ArcA overexpression suppresses the TCA cycle, causing the overflow of AcCoA, a precursor of MVA. We predicted the xylose content that maximizes the MVA production. This approach demonstrates the possibility of a great progress in the computer-aided rational design of the microbial cell factories for useful metabolite production.

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Section: Introductionmentioning

confidence: 99%

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

Matsuoka

Kurata

2020

Front. Bioeng. Biotechnol.

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“…The functioning of down-regulation of rplW (encoding 50S ribosomal subunit protein L23), tyrU (encoding tRNA-Tyr) and rnpB (encoding M1 RNA, catalytic component of tRNA processing enzyme RNase P) was associated with the inhibition of protein synthesis, which increased partitioning of total carbon to lipids 8 . Up-regulation of nnr , facilitating the repair of NAD(P)H hydrates to NAD(P)H 39 , could be an important complement strategy in enhancing NADPH availability, which plays an important role in FFAs production 19,40 . In all, these findings provide new insights into linkages between cell functions and product biosynthesis, guiding avenues for further strain development.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids inEscherichia coli

Fang

Fan

Wang

et al. 2020

Preprint

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18To construct a superior microbial cell factory for chemical synthesis, a major 19 challenge is to fully exploit cell potential via identifying and engineering beneficial 20 gene targets in the sophisticated metabolic networks. Here, we develop an approach 21 that integrates CRISPR interference (CRISPRi) to readily modulate genes expression 22 and omics analyses to identify potential targets in multiple cellular processes, 23 enabling systematical discovery of beneficial chromosomal gene targets that can be 24 engineered to optimize free fatty acids (FFAs) production in Escherichia coli. We 25 identify 56 beneficial genes via synergistic CRISPRi-Omics strategy, including 46 26 novel targets functioning in cell structure and division, and signaling transduction that 27 efficiently facilitate FFAs production. Upon repressing ihfA and overexpressing aidB 28 and tesA' in E. coli, the recombinant strain LihfA-OaidB results in a FFAs titer of 29 21.6 g L -1 in fed-batch fermentation, which, to our best knowledge, is the maximum 30 FFAs titer by the recombinant E. coli reported to date. 31 3

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“…We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13 C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy).…”

Section: Resultsmentioning

confidence: 99%

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

Sui

Schütze

Ouyang

et al. 2020

Preprint

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Background: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH regeneration enzymes under the control of Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the reverse TCA cycle ( maeA gene) followed by engineering the flux through the pentose phosphate pathway ( gndA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

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Effect of NADPH availability on free fatty acid production in Escherichia coli

Cited by 38 publications

References 32 publications

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids inEscherichia coli

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

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