Machine learning model of the catalytic efficiency and specificity of acyl-ACP thioesterase variants generated by natural and in vitro directed evolution

Modulating the catalytic activity of acyl-ACP thioesterase (TE) is an important biotechnological target for effectively increasing flux and diversifying products of the fatty acid biosynthesis pathway. In this study, a directed evolution approach was developed to improve the fatty acid productivity and fatty acid diversity of E. coli strains expressing variant acyl-ACP TEs. A single round of directed in vitro evolution, coupled with a high-throughput colorimetric screen, identified 26 novel acyl-ACP TE variants, which convey up to 10-fold increase in fatty acid productivity, and altered fatty acid profiles when expressed in a bacterial host strain. These in vitro generated variant acyl-ACP TEs, in combination with 31 natural variants isolated from diverse phylogenetic origins were analyzed with a random forest classifier machine learning tool, generating a quantitative model that identified 22 amino acid residues, which define important structural features that determine the substrate specificity of acyl-ACP TE.

Highly Efficient Production of D-xylonic Acid From Corn Stover Hydrolysate in Gluconobacter Oxydans By mGDH Overexpression

Background: D-xylonic acid is a versatile platform chemical with wide potential applications as the water reducer and disperser for cement and the precursor for 1,4-butanediol and 1,2,4-tributantriol. Microbial production of D-xylonic acid with bacteria like Gluconobacter oxydans from cheap lignocellulosic feedstock is generally regarded as one of the most promising and cost-effective methods to industrial production, but xylonic acid productivity is reduced by high substrate inhibition and hydrolysate inhibitors. Results: D-xylonic acid productivity of G. oxydans DSM2003 was improved by overexpressing the mGDH gene, which encoded the membrane-bound glucose dehydrogenase. Using the mutated plasmids based on pBBR1MCS-5 in our previous work, the recombinant strain G. oxydans/pBBR-R3510-mGDH with the significant improvement in D-xylonic acid production and the strengthened tolerance to hydrolysate inhibitors was obtained. The fed-batch biotransformation of D-xylose by this recombinant strain reached the record high titer (588.7 g/L), yield (99.4%), and space-time yield (8.72 g/L/h). Moreover, up to 246.4 g/L D-xylonic acid was produced directly from corn stover hydroxylates without detoxification at a yield of 98.9% and a space-time yield of 11.2 g/L/h. In addition, G. oxydans/pBBR-R3510-mGDH performed strong tolerance to typical inhibitors, i.e., formic acid, furfural, and 5-hydroxymethylfurfural. Conclusion: Through overexpression of mgdh in G. oxydans, we obtained a recombinant strain G. oxydans/pBBR-R3510-mGDH. It was capable to efficiently produce xylonic acid from the corn stover hydrolysate under high concentrations of inhibitors. The high D-xylonic acid productivity made G. oxydans/pBBR-R3510-mGDH an attractive choice for biotechnical production.

Semicontinuous Fermentation of Cassava by Streptococcus bovis Using Membrane Bioreactor to Produce Lactic Acid

The main objective of this study is to compare the production of lactic acid from cassava by Streptococcus bovis in semicontinuous system with and without membrane separation. Fermentation was run for 72 h, and membrane filtration was performed using micro filtration membrane (MF). The results obtained demonstrate that higher productivity of lactic acid was achieved using fermentation coupled with membrane separation. It was also found that dilution rate (D) of 0.02 h-1 led to slight increase in the lactic acid productivity. Despite this small increase, this investigation shows that membrane separation has the potential to improve the performance of semicontinuous fermentation system.