OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate

Ghosh, Indro Neil; Martien, Julia; Hebert, Alexander S.; Zhang, Yaoping; Coon, Joshua J.; Amador‐Noguez, Daniel; Landick, Robert

doi:10.1016/j.ymben.2018.12.008

Cited by 34 publications

(41 citation statements)

References 85 publications

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“…Lysis and digestion were performed as previously described (Ghosh et al, 2019). Briefly, the cell pellets were first re-suspended in 1 mL 6 M guanidine hydrochloride (GnHCl) to lyse the cells and precipitate the protein, MeOH was added to 90% final concentration.…”

Section: Methodsmentioning

confidence: 99%

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

et al. 2019

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Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.

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Section: Methodsmentioning

confidence: 99%

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

et al. 2019

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“…For example, Atsumi et al engineered E. coli to produce isobutanol up to 22 g/L in shake flasks [33,42], which was further increased to 50 g/L in bioreactors during batch cultures with in situ product removal by gas stripping [43]. Recently, Ghosh and co-workers optimized their constructs based on models to predict the impacts of enzyme synthesis cost on cellular growth rates and obtained a recombinant strain with an isobutanol productivity of 3 g/h/g DCW (dry ell weight) [44]. Li and co-workers found that B. subtilis was a feasible host because it had a higher isobutanol tolerance than E. coli and C. glutamicum and produced up to 2.62 g/L isobutanol during fed-batch fermentation [36].…”

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confidence: 99%

“…Despite of significant progress achieved on isobutanol production in various microorganisms, most require aerobic fermentation conditions, a costly input for industrial production. In addition, current efforts on improving isobutanol production in these microorganisms have been focused on increasing the pyruvate pool, eliminating by-product formation, and maintaining redox balance [44,[47][48][49][50][51]. For example, the Entner-Doudoroff (ED) pathway was applied into E. coli, and the ED pathway-dependent isobutanol-producing E. coli strain was further optimized to produce 15.0 g/L isobutanol after byproduct biosynthesis genes were inactivated [52].…”

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Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Qiu

Shen

Yan

et al. 2020

Biotechnol Biofuels

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Background: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. Results: In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. Conclusions: This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

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“…The resulting E. coli strain, ED02, produced 13.67 g/L isobutanol with a productivity of 0.456 g/L/h, a 56.8 and 88.1% improvement over the parent strain, respectively. Ghosh et al (2019) used the Optimization by Selection and Sequencing (OptSSeq) strategy to regulate expression levels of genes in the isobutanol synthetic pathway. They found that the optimum levels of pathway enzymes (AlsS, IlvC, IlvD, Kivd, and AdhA) were a molar ratio of 2.5:6.7:2:1:5.2, which led to 3 g/h/gDCW of isobutanol production.…”

Section: C3-c4 Alcohol Tolerance and Production In E Colimentioning

confidence: 99%

Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2–C6 Bioalcohol Production

Liang

Liu

Freed

et al. 2020

Front. Bioeng. Biotechnol.

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Biofuel production from renewable and sustainable resources is playing an increasingly important role within the fuel industry. Among biofuels, bioethanol has been most widely used as an additive for gasoline. Higher alcohols can be blended at a higher volume compared to ethanol and generate lower greenhouse gas (GHG) emissions without a need to change current fuel infrastructures. Thus, these fuels have the potential to replace fossil fuels in support of more environmentally friendly processes. This review summarizes the efforts to enhance bioalcohol production in engineered Escherichia coli over the last 5 years and analyzes the current challenges for increasing productivities for industrial applications.

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OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate

Cited by 34 publications

References 85 publications

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2–C6 Bioalcohol Production

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